CD73 inhibitors and pharmaceutical uses thereof

ABSTRACT

CD73 (also known as ecto-5′-nucleotidase) inhibitor compounds are provided, as well as compositions and uses thereof for treating or preventing CD73-associated or related diseases, disorders and conditions, including cancer- and immune-related disorders. CD73 inhibitor compounds include compounds having the structure set forth in Formula I and pharmaceutically acceptable esters or salts thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 16/566,327, filed Sep. 10, 2019, which claims the benefit ofpriority from U.S. Provisional Application No. 62/773,267, filed Nov.30, 2018, and Chinese Patent Application No. 201811057145.X, filed Sep.11, 2018, each of which is hereby incorporated by reference in itsentirety. This application also claims the benefit of priority fromChinese Patent Application No. 202011346141.0, filed Nov. 25, 2020,which is hereby incorporated by reference in its entirety.

FIELD

The present disclosure relates to compounds and compositions thatinhibit CD73 (ecto-5′-nucleotidase), and uses thereof for treatingand/or preventing CD73-associated or related diseases, disorders andconditions, including cancer- and immune-related disorders.

BACKGROUND

Ecto-nucleotidases are a group of cell-surface located ecto-enzymes. Themembers of the ecto-nucleotidase family include ecto-nucleotidepyrophosphatase/phosphodiesterases (E-NPPs), ecto-nucleosidetriphosphate diphosphohydrolases (E-NTPDases), ecto-5′-nucleotidase(e5NT, also known as CD73) and alkaline phosphatase (AP). These enzymeshydrolyze a variety of extracellular nucleotides to nucleosidesincluding adenosine. Extracellular nucleotides are important signalingmolecules that trigger cellular responses by acting on their respectivereceptors (for example, adenosine activates P1 receptors, andnucleotides thereof (ADP, ATP) activate P2 receptors). Adenosine5′-monophosphate (AMP) is a major substrate of CD73 that is hydrolyzedto adenosine. Adenosine is ubiquitously present in the body and is animportant regulator of purinergic cell signaling that is vital for manyphysiological and pathophysiological processes.

There is a wealth of data implicating CD73 enzymatic activity inpromotion and metastasis of cancer. CD73 is up-regulated in many cancercell-types and tumors, and its expression has been shown to beassociated with tumor neovascularization, invasiveness and metastasis.The hydrolytic cascade from extracellular ATP to adenosine is animportant immunosuppressive regulatory pathway in the tumormicroenvironment. CD73 overexpression impairs adaptive antitumor immuneresponses, and enhances tumor growth and metastasis. Extracellularadenosine is also implicated in regulating adaptive responses tohypoxia. Decreasing e5NT activity with monoclonal antibodies, siRNA, andsmall molecule inhibitors including AMPCP (adenosine [(α,β)-methylene]diphosphate) has been shown to attenuate the growth and metastasis oftumors (see, e.g., Zhou et al., Oncol. Rep. 17 (2007): 1341-1346; Staggand Smyth, Oncogene, 29 (2010): 5346-5358). Tumor growth is alsoimpaired in CD73-deficient mice and it has been established that theseeffects are largely mediated by diminished adenosine production in thesemice. Inhibitors of CD73 have thus been actively explored for theirtherapeutic potential against cancer (see, e.g., M. al-Rashida et al.,Eur. J. Med. Chem., 115 (2016): 484-494, and references cited therein).

Tumor cells overcome anti-tumor responses in part throughimmunosuppressive mechanisms. There are several such immune modulatorymechanisms. Among them, adenosine is a key factor which can be generatedby both cancer and immune cells in the tumor microenvironment tosuppress anti-tumor responses. The generation of adenosine fromadenosine triphosphate (ATP) is catalyzed by two cell-surface proteins,CD73 and CD39, and can be enhanced under metabolic stress, such as tumorhypoxic conditions. Adenosine exerts its immune-regulatory functionsthrough four adenosine receptors (ARs), called A1, A2A, A2B, and A3,which are expressed on various immune cells. Overexpression ofadenosine-generating enzymes such as CD73 and ARs has been correlatedwith tumor progression in a multitude of cancer types. Since thesignaling of ARs enhances tumor progression, their modulation alsorepresents a promising therapeutic approach for cancer (M. H. Kazemi, etal., J. Cell. Physiol., 233 (2018): 2032-2057, and references citedtherein).

As mentioned above, ecto-nucleotidases are cell surface-located enzymesthat regulate purinergic (and pyrimidinergic) signaling pathways. Thereare four distinct families of ecto-nucleotidases: ecto-nucleosidetriphosphate diphosphohydrolasea (CD39), ecto-nucleotidepyrophosphatases/phosphodiesterases, alkaline phosphatases, andecto-5′-nucleotidase (e5NT, also known as CD73). CD73 is aglycophosphatidylinositol-anchored di-Zn metallophosphatase. CD73catalyzes the dephosphorylation of extracellular adenosine monophosphate(AMP) to adenosine. This ecto-enzymatic cascade in tandem with CD39generates adenosine from ATP. The CD73-catalyzed conversion of AMP toadenosine is considered to be a major contributor to the elevated levelsof extracellular adenosine in the tumor microenvironment (Stagg, J. etal., Proc. Natl. Acad. Sci. USA.: 107 (2010): 1547-1552). Expression ofCD73 is directly upregulated by the hypoxia-inducible factor-1α, whichexplains the observed increase in extracellular adenosine in hypoxicmalignant tumors. CD73 is also expressed by T-regulatory cells (Tregs)and promotes Treg-mediated immunosuppression (Stagg J, et al., CancerRes. 71 (2011): 2892-2900). In addition, CD73 is induced by transforminggrowth factor-β (TGF-β), tumor necrosis factor-α (TNF-α), hepatocytegrowth factor (HGF), interleukin-6 (IL-6), mitogen-activated proteinkinase (MAPK), signal transducers and activators of transcription 3(STAT3), interleukin-2 (IL-2), retinoic acid, int/wingless (WNT),epithelial-to-mesenchymal transition, and p53 mutations. CD73 isoverexpressed in a multitude of tumor types and promotes the invasion,migration, and adhesion of tumor cells. CD73 is also associated withimmune tolerance and poor prognosis in cancer. CD73 is thus a promisingtarget for the development of anti-cancer drugs. Furthermore, CD73inhibitors have potential for the treatment of other diseases mediatedby adenosine and its receptors (Y.-P. Gong, et al., Expert Opin. Ther.Pat., 28 (2018): 167-171).

The adenosine pathway is also known to be a major immunosuppressivecomponent of many human tumors (for review, see Whiteside, T. L., ExpertRev. Anticancer Ther., 17 (2017): 527-535). Adenosine and inosine emergeas critical immune checkpoints in cancer. Cooperation of the adenosineand PGE2 pathways in the tumor microenvironment contributes tosuppression of anti-tumor immune effector cells. Targeting of theadenosine pathway with pharmacologic inhibitors or antibodies is thus apromising therapeutic strategy in cancer.

Blocking activities of ecto-nucleotidases or of adenosine receptorsignaling in preclinical in vivo studies has been successful ininhibiting tumor growth and metastasis. The adenosine pathway blockadealone or in combination with other immune therapies, includingcheckpoint inhibitors, is currently being implemented in initial phase Iclinical trials for subjects with advanced malignancies.

Small-molecule inhibitors of CD73 have been reported. For example, Adamset al. (International PCT Application Publication No. WO2017/098421)describe substituted benzothiadiazine derivatives that are inhibitors ofCD73, pharmaceutical compositions thereof, and their use in thetreatment of cancer, pre-cancerous syndromes and diseases associatedwith CD73 inhibition.

Debien et al. (International PCT Application Publication No.WO2017/120508; U.S. Patent Application Publication No. US2017/0267710)describe compounds that modulate the conversion of AMP to adenosine by5′-nucleotidase, ecto, compositions containing the compounds, methodsfor synthesizing the compounds, and the use of such compounds andcompositions for the treatment and/or prevention of a diverse array ofdiseases that are mediated by 5′-nucleotidase, ecto.

Cacatian et al. (International PCT Application Publication No.WO2015/164573) describe purine derivatives and pharmaceuticalcompositions thereof which are inhibitors of CD73 and are useful in thetreatment of cancer.

Chen et al. (International PCT Application Publication No. WO2018/049145) disclose preparation of nucleotides as ectonucleotidaseinhibitors, and the use of the compounds in treating or preventingcancer.

The contents of all documents and references cited herein are herebyincorporated by reference in their entirety.

SUMMARY

The present disclosure relates to compounds and compositions comprisingthe compounds that inhibit the activity of ecto-5′-nucleotidase (alsoknown as e5NT, CD73, NT5E, and 5NT). Inhibition of CD73 enzymaticactivity leads to inhibition or modulation of extracellular adenosinelevels and thus modulates the physiological environment of cells andtissues.

The present disclosure also relates to the use of such compounds andcompositions for the treatment and/or prevention of diseases, disordersand conditions mediated, in whole or in part, by CD73. CD73 inhibitorshave been linked to the treatment of many disorders, including cancer,fibrosis, neurological and neurodegenerative disorders (e.g., depressionand Parkinson's disease), cerebral and cardiac ischemic diseases,immune-related disorders, and disorders with an inflammatory component.In particular embodiments, the CD73 inhibitor compounds and compositionsdescribed herein can act to inhibit the immunosuppressive activityand/or the anti-inflammatory activity of CD73, and are useful astherapeutics or prophylactic therapies when such inhibition isdesirable.

In a first broad aspect, there are provided compounds of Formula I andpharmaceutically acceptable salts or esters thereof.

where W is oxygen, sulfur, nitrogen, or a methylene group; X is a moietyselected from phosphonyl (—P(═O)(OR)—), sulfonyl (—S(═O)₂—), andcarbonyl (—C(═O)—) fragment, where R is hydrogen, ester-forming group,or protecting group; or X and W together form the —(CR⁷R⁸)_(n)—, where nis an integer from 0 to 3, and R⁷ and R⁸ are independently selected froma hydrogen, a halogen, a hydroxyl group and a lower alkyl group having 1to 4 carbon atoms; Y is selected from phosphonate (—PO₃R₂), sulfonate(—SO₃R), and carboxylate (—CO₂R), where R is a hydrogen, anester-forming group, or a protecting group; R¹ is a hydroxyl group or ahydrogen; R² is chlorine or a hydrogen; and R³ and R⁴ are independentlyselected from hydrogen, alkyl group, alkenyl group and alkynyl group,where at least one of R³ and R⁴ has a carbon number of from 1 to 30,such as without limitation from 1 to 10, from 11 to 20, from 11 to 30,or from 21 to 30, and wherein, when W is O or S, the carbon number isnot 1 to 10 (i.e., when W is O or S, at least one of R³ and R⁴ has acarbon number of from 11 to 30).

In one embodiment, R³, R⁴, and the nitrogen atom to which they areattached form a heterocyclic system which is independently selected froma monocycle, a bicycle, a tricycle, a spiral-ring, a fused-ring, or abridged-ring system.

In one embodiment, R³ and R⁴ are independently selected from hydrogenand a ring system, the ring system being a monocycle, bicycle, tricycle,spiral-ring, fused-ring or bridged-ring containing carbocyclic (aromaticor non-aromatic) or heterocyclic ring system, and the ring system beingsubstituted or non-substituted, provided that R³ and R⁴ are not bothhydrogen at the same time, and provided that the ring system is not amonocycle when W is O or S.

In a further embodiment, R³ is hydrogen or a lower alkyl (e.g., C₁₋₆)and R⁴ is —C(═O)R⁵, —C(═O)NHR⁵ or —C(═O)OR⁵, where R⁵ is a C₁₋₃₀ alkylgroup, a C₂₋₃₀ alkenyl group or a C₂₋₃₀ alkynyl group, wherein the C₁₋₁₀and C₂₋₁₀ groups are excluded when W is O or S (i.e., R⁵ is a C₁₋₃₀alkyl, C₁₋₃₀ alkenyl or C₁₁₋₃₀ alkynyl group when W is O or S).

In some embodiments, R³ is hydrogen or a lower alkyl and R⁴ is —C(═O)R⁵,—C(═O)NHR⁵ or —C(═O)OR⁵, where R⁵ is a ring system which is a monocycle,bicycle, tricycle, spiral-ring, fused-ring or bridged-ring containing acarbocyclic (aromatic or non-aromatic) or heterocyclic ring system, thecarbocyclic or heterocyclic ring system being substituted ornon-substituted, wherein the R⁵ ring system is not a monocycle when W isO or S.

In one embodiment, there are provided compounds of Formula II and/orFormula III, and pharmaceutically acceptable salts or esters thereof:

where W, X, Y, R¹, R³, and R⁴ are as defined above.

In another embodiment, there are provided compounds of Formula IV, andpharmaceutically acceptable salts or esters thereof:

where X, Y, R¹, R², R³, and R⁴ are as defined above.

In another embodiment, there are provided compounds of Formula IVa, andpharmaceutically acceptable salts or esters thereof:

where X, Y, R¹, R², R³, and R⁴ are as defined above.

In another embodiment, there are provided compounds of Formula IVb, andpharmaceutically acceptable salts or esters thereof:

where X, Y, R¹, R², R³, and R⁴ are as defined above.

In another embodiment, there are provided compounds of Formula IVc, andpharmaceutically acceptable salts or esters thereof:

where n, X, Y, R¹, R², R³, and R⁴ are as defined above.

In yet another embodiment, there are provided compounds of Formulae Vand/or VI, and pharmaceutically acceptable salts or esters thereof:

where X, Y, R¹, R³, and R⁴ are as defined above.

In another embodiment, there are provided compounds of Formulae Vaand/or VIa, and pharmaceutically acceptable salts or esters thereof:

where X, Y, R¹, R³, and R⁴ are as defined above.

In another embodiment, there are provided compounds of Formulae VIIand/or VIII, and pharmaceutically acceptable salts or esters thereof:

where R is hydrogen, an ester-forming group, or a protecting group; andR¹, R², R³, and R⁴ are as defined above.

In another embodiment, there are provided compounds of Formulae VIIaand/or VIIIa, and pharmaceutically acceptable salts or esters thereof:

where R is hydrogen, an ester-forming group, or a protecting group; andR¹, R², R³, and R⁴ are as defined above.

In yet another embodiment, there are provided compounds of Formulae IX,and pharmaceutically acceptable salts or esters thereof:

where R is hydrogen, an ester-forming group, or a protecting group; andR¹, R², R³, and R⁴ are as defined above.

In another embodiment, there are provided compounds of Formulae IXa, andpharmaceutically acceptable salts or esters thereof:

where R is hydrogen, an ester-forming group, or a protecting group; andR¹, R², R³, and R⁴ are as defined above.

In one embodiment, R¹ is a hydroxyl group (i.e., the carbohydrate moietyin the compound is a D-ribosyl moiety). In another embodiment, R¹ ishydrogen (i.e., the carbohydrate moiety in the compound is a2-deoxy-D-ribosyl moiety).

In another embodiment, R² is hydrogen. In yet another embodiment, R² ishydrogen and R¹ is a hydroxyl group (i.e., the compound is an adenosinederivative). In another embodiment, R² is hydrogen and R¹ is hydrogen(i.e., the compound is a deoxyadenosine derivative). In still anotherembodiment, R² is hydrogen and both R³ and R⁴ are not hydrogen (i.e.,the compound is an adenosine derivative or a deoxyadenosine derivativewith substituent groups on the amino group of the adenine moiety). Inanother embodiment, R² is chlorine and the compound is a2-chloro-D-adenosine derivative or a 2-chloro-D-deoxyadenosinederivative.

In some embodiments, R³ is hydrogen or a lower alkyl (e.g., C₁₋₆), andR⁴ is an alkyl, alkenyl, or alkynyl group having 1 to 30 carbon atoms(i.e., a C₁₋₃₀ alkyl group, a C₂₋₃₀ alkenyl group, or a C₂₋₃₀ alkynylgroup) and R⁴ has 11 to 30 carbon atoms when W is O or S. In someembodiments, R³ is a hydrogen or a lower alkyl, and R⁴ is a groupcontaining an adamantyl moiety. R⁴ may be, for example, substituted ornon-substituted 1-adamantyl, substituted or non-substituted 2-adamantyl,substituted or non-substituted 1-adamantylmethyl, substituted ornon-substituted 1-adamantylethyl, substituted or non-substituted1-adamantylpropyl, or substituted or non-substituted 1-adamantylbutyl.In some embodiments, R³ is a hydrogen or a lower alkyl, and R⁴ is agroup containing a naphthyl moiety. R⁴ may be, for example, substitutedor non-substituted α-naphthyl, substituted or non-substitutedβ-naphthyl, substituted or non-substituted α-naphthylmethyl, substitutedor non-substituted β-naphthylmethyl, substituted or non-substitutednaphthylethyl, substituted or non-substituted naphthylpropyl, orsubstituted or non-substituted naphthylbutyl.

In another embodiment, R³ is hydrogen or a lower alkyl, and R⁴ is asubstituent group containing a monocycle, bicyclic, tricyclic, ormulticyclic ring system, where the ring system is fused, spiral,bridged, or parallel, and where the ring system is carbocyclic,aliphatic, aromatic, heterocyclic, or a combination thereof.

In a further embodiment, R³ is hydrogen or a lower alkyl, and R⁴ is—C(═O)R⁵, —C(═O)NHR⁵ or —C(═O)OR⁵, where R⁵ is an alkyl group or analkenyl group or an alkynyl group having 1 to 30 carbon atoms, whereinC₁ to C₁₀ groups are excluded when W is O or S.

In some embodiments, R³ is hydrogen or a lower alkyl, and R⁴ is—C(═O)R⁵, —C(═O)NHR⁵ or —C(═O)OR⁵, where R⁵ is a substituent groupcontaining a monocyclic, bicyclic, tricyclic, or multicyclic ringsystem, where the ring system is fused, spiral, bridged, or parallel,and where the ring system is carbocyclic, aliphatic, aromatic,heterocyclic, or a combination thereof, wherein R is not a monocyclicring system when W is O or S.

In one embodiment, R⁴ is a group containing an adamantyl moiety. Infurther embodiment, R⁴ is a substituted or non-substituted 1-adamantylor 2-adamantyl. In yet another embodiment, R⁴ is a substituted ornon-substituted 1-adamantylmethyl. In some embodiments, R⁴ is1-adamantylethyl, 1-adamantylpropyl, or 1-adamantylbutyl, where theadamantyl moiety can be substituted or non-substituted.

In another embodiment, R⁴ is a group containing naphthyl moiety. Infurther embodiment, R⁴ is substituted or non-substituted α-naphthy orβ-naphthyl. In other embodiment, R⁴ is α-naphthylmethyl orβ-naphthylmethyl, without or with further substitution. In yet anotherembodiment, R⁴ is selected from naphthylethyl, naphthylpropyl, andnaphthylbutyl, where the naphthyl moiety can be non-substituted orsubstituted.

In another embodiment, R³ and R⁴, together with the nitrogen to whichthey are attached, form a tricyclic fused ring system, such as withoutlimitation a substituted or unsubstituted carbazolyl moiety.

In another embodiment, R³ and R⁴, together with the nitrogen to whichthey are attached, form a heterocyclic system independently selectedfrom monocycle, bicycle, tricycle, spiral-ring, fused-ring, andbridged-ring.

In some embodiments, there are provided compounds of Table 1 andpharmaceutically acceptable salts or esters thereof.

TABLE 1 Structures of example compounds. Compound No. Structure  1

 2

 3

 4

 5

 6

 7

 8

 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

In some embodiments, there are provided compounds of Table 1a andpharmaceutically acceptable salts or esters thereof.

TABLE 1a Structures of example compounds. Compound Number Structure a-1 

a-2 

a-3 

a-4 

a-5 

a-6 

a-7 

a-8 

a-9 

a-10

a-11

a-12

a-13

a-14

a-15

a-16

a-17

a-18

a-19

a-20

a-21

a-22

a-23

a-24

a-25

a-26

a-27

a-28

a-29

a-30

a-31

a-32

a-33

a-34

a-35

a-36

a-37

a-38

a-39

a-40

a-41

a-42

a-43

a-44

a-45

a-46

a-47

a-48

a-49

a-50

a-51

a-52

a-53

a-54

a-55

a-56

a-57

a-58

a-59

a-60

a-61

In some embodiments, there are provided compounds of Table 1b andpharmaceutically acceptable salts or esters thereof.

TABLE 1b Structures of example compounds. Compound Number Structure b-1 

b-2 

b-3 

b-4 

b-5 

b-6 

b-7 

b-8 

b-9 

b-10

b-11

b-12

b-13

b-14

b-15

b-16

b-17

b-18

b-19

b-20

b-21

b-22

b-23

b-24

b-25

b-26

b-27

b-28

b-29

b-30

b-31

b-32

b-33

b-34

b-35

b-36

b-37

b-38

b-39

b-40

b-41

b-42

b-43

b-44

b-45

b-46

b-47

b-48

b-49

b-50

b-51

b-52

b-53

b-54

b-55

b-56

b-57

b-58

b-59

b-60

b-61

In some embodiments, there are provided compounds of Table 1c andpharmaceutically acceptable salts or esters thereof.

TABLE 1c Structures of example compounds. Compound Number Structure c-1 

c-2 

c-3 

c-4 

c-5 

c-6 

c-7 

c-8 

c-9 

c-10

c-11

c-12

c-13

c-14

c-15

c-16

c-17

c-18

c-19

c-20

c-21

c-22

c-23

c-24

c-25

c-26

c-27

c-28

c-29

c-30

c-31

c-32

c-33

c-34

c-35

c-36

c-37

c-38

c-39

c-40

c-41

c-42

c-43

c-44

c-45

c-46

c-47

c-48

c-49

c-50

c-51

c-52

c-53

c-54

c-55

c-56

c-57

c-58

c-59

c-60

c-61

c-62

c-63

c-64

c-65

c-66

c-67

c-68

c-69

c-70

c-71

c-72

c-73

c-74

c-75

c-76

c-77

c-78

c-79

In some embodiments, there is provided a compound as described hereinwherein the C, H, O, and N atoms in the compound are each independentlyselected from atoms of natural abundance and isotope-enriched atoms.Examples of isotope-enriched atoms include, without limitation, ¹²C,¹³C, and ¹⁴C for carbon; ¹H, ²H, and ³H for hydrogen; ¹⁶O, ¹⁷O, and ¹⁸Ofor oxygen; and ¹⁴N and ¹⁵N for nitrogen.

In a second broad aspect, there are provided pharmaceutical compositionscomprising a compound described herein, or a pharmaceutically acceptablesalt or ester thereof, and a pharmaceutically acceptable carrier. Insome embodiments, there are provided pharmaceutical compositionscomprising a compound of any one of Formulae I to IX, or apharmaceutically acceptable salt or ester thereof, and apharmaceutically acceptable carrier. In some embodiments, there areprovided pharmaceutical compositions comprising a compound of any one ofFormulae I to IX, or a pharmaceutically acceptable salt or esterthereof, wherein, in the compound, one of R³ and R⁴ is not hydrogen or aC₁ to C₁₀ alkyl alkenyl, or alkynyl group when W is O or S. In someembodiments, there are provided pharmaceutical compositions comprising acompound shown in Tables 1, 1a, 1b, and 1c, or a pharmaceuticallyacceptable salt or ester, and a pharmaceutically acceptable carrier. Insome embodiments, the pharmaceutically acceptable carrier comprises acream, an emulsion, a gel, a liposome, or a nanoparticle.

In some embodiments, the pharmaceutically acceptable carrier furthercomprises at least one additional therapeutic agent, such as, withoutlimitation, a chemotherapeutic agent, an immune- and/orinflammation-modulating agent, an anti-hypercholesterolemia agent, or ananti-infective agent. In an embodiment, the at least one additionaltherapeutic agent is an immune checkpoint inhibitor. Non-limitingexamples of immune checkpoint inhibitors include ipulimumab, nivolumaband lambrolizumab.

In a third broad aspect, there are provided compounds, compositions, andmethods of inhibiting CD73 activity in a subject in need thereofcomprising administering to the subject an effective amount of acompound and/or a pharmaceutical composition described herein.

In particular embodiments, the compounds described herein act to inhibitthe immunosuppressive activity and/or the anti-inflammatory activity ofCD73, and are useful as therapeutic or prophylactic therapy when suchinhibition is desired. Unless otherwise indicated, when uses of thecompounds of the present invention are described herein, it is to beunderstood that such compounds may be in the form of a composition(e.g., a pharmaceutical composition). As used herein, the terms “CD73inhibitor”, “CD73 blocker”, “adenosine by ecto-5′-nucleotidaseinhibitor”, “NT5E inhibitor”, “5NT inhibitor” and all other relatedart-accepted terms are used interchangeably to refer to a compoundcapable of inhibiting, either directly or indirectly, the CD73 receptorin an in vitro assay, an in vivo model, and/or other assay meansindicative of CD73 inhibition and potential therapeutic or prophylacticefficacy. The terms also refer to compounds that exhibit at least sometherapeutic or prophylactic benefit in a human subject.

Although the compounds of the present invention are believed to haveeffect by inhibition of CD73, a precise understanding of the compounds'underlying mechanism of action is not required to practice theinvention. For example, the compounds may also have effect, at least inpart, through modulation (e.g., inhibition) of other components of thepurinergic signaling pathway (e.g., CD39). The purinergic signalingsystem consists of transporters, enzymes and receptors responsible forthe synthesis, release, action, and extracellular inactivation of(primarily) ATP and its extracellular breakdown product adenosine.Because inhibition of CD73 results in decreased adenosine production,CD73 inhibitors can be used for the treatment of diseases or disordersmediated by adenosine and its actions on adenosine receptors, includingA1, A2A, A2B and A3.

For purposes of the present disclosure, the purinergic signaling processcan be described as comprising the following components. The purinergicreceptors (P1, P2X and P2Y), a first component, are membrane receptorsthat mediate various physiological functions (e.g., relaxation of gutsmooth muscle) as a response to the release of ATP or adenosine; ingeneral, all cells have the ability to release nucleotides into theextracellular environment, frequently through regulated exocytosis. Thenucleoside transporters (NTs), a second component, are membranetransport proteins which transport nucleoside substrates (e.g.,adenosine) across cell membranes; the extracellular concentration ofadenosine can be regulated by NTs, possibly in the form of a feedbackloop connecting receptor signaling with transporter function. Aspreviously described, the ecto-nucleotidases (CD73 and CD39) hydrolyzenucleotides released into the extracellular environment and comprise afurther component.

In some embodiments, there are provided methods for treating orpreventing cancer in a subject (e.g., a human) comprising administeringto the subject a therapeutically effective amount of at least one CD73inhibitor compound or composition described herein. In some embodimentsof such methods, the subject is administered at least one CD73 inhibitorcompound or composition in an amount effective to reverse, slow or stopthe progression of CD73-mediated immunosuppression. In some embodiments,the CD73-mediated immunosuppression is mediated by an antigen-presentingcell (APC).

The type of cancer or tumor that can be treated or prevented using thecompounds and compositions described herein is not meant to beparticularly limited. Examples of cancers and tumors that can be treatedor prevented using the compounds and compositions described hereininclude, but are not limited to: cancers of the prostate, colorectum,pancreas, cervix, stomach, endometrium, brain, liver, bladder, ovary,testis, head, neck, skin (including melanoma and basal carcinoma),mesothelial lining, white blood cell (including lymphoma and leukemia),esophagus, breast, muscle, connective tissue, lung (including small-celllung carcinoma and non-small-cell carcinoma), adrenal gland, thyroid,kidney, bone, glioblastoma, mesothelioma, renal cell carcinoma, gastriccarcinoma, sarcoma, choriocarcinoma, cutaneous basocellular carcinoma,and testicular seminoma. In some embodiments of the present invention,the cancer is melanoma, colon cancer, pancreatic cancer, breast cancer,prostate cancer, lung cancer, leukemia, a brain tumor, lymphoma,sarcoma, ovarian cancer, or Kaposi's sarcoma.

In some embodiments, there are provided methods of treating a subjectreceiving a bone marrow transplant or peripheral blood stem celltransplant by administering a therapeutically effective amount of anCD73 inhibitor compound or composition sufficient to increase thedelayed-type hypersensitivity reaction to tumor antigen, delay thetime-to-relapse of post-transplant malignancy, increase relapse-freesurvival time post-transplant, and/or increase long-term post-transplantsurvival.

In certain embodiments, there are provided methods for treating orpreventing an infective disorder (e.g., a viral infection) in a subject(e.g., a human) comprising administering to the subject atherapeutically effective amount of at least one CD73 inhibitor compoundor composition provided herein. In some embodiments, the infectivedisorder is a viral infection (e.g., a chronic viral infection), abacterial infection, a fungal infection, or a parasitic infection. Incertain embodiments, the viral infection is human immunodeficiency virusor cytomegalovirus.

In still other embodiments, there are provided methods for treatingand/or preventing immune-related diseases, disorders and conditions;diseases having an inflammatory component; as well as disordersassociated with the foregoing; with at least one CD73 inhibitor compoundor composition provided herein.

Other diseases, disorders and conditions that can be treated orprevented, in whole or in part, by inhibition of CD73 activity arecandidate indications for the CD73 inhibitor compounds and compositionsprovided herein.

In some embodiments, there is further provided the use of the CD73inhibitor compounds and compositions described herein in combinationwith one or more additional agents. The one or more additional agentsmay have some CD73-modulating activity and/or they may function throughdistinct mechanisms of action. In some embodiments, such agents compriseradiation (e.g., localized radiation therapy or total body radiationtherapy) and/or other treatment modalities of a non-pharmacologicalnature. When combination therapy is utilized, the CD73 inhibitor(s) andone additional agent(s) may be in the form of a single composition ormultiple compositions, and the treatment modalities can be administeredconcurrently, sequentially, or through some other regimen. By way ofexample, in some embodiments there is provided a treatment regimenwherein a radiation phase is followed by a chemotherapeutic phase. Acombination therapy can have an additive or synergistic effect.

In some embodiments, there is provided the use of a CD73 inhibitorcompound or composition described herein in combination with bone marrowtransplantation, peripheral blood stem cell transplantation, or othertypes of transplantation therapy.

In particular embodiments, there is provided the use of the inhibitorsof CD73 function described herein in combination with immune checkpointinhibitors. The blockade of immune checkpoints, which results in theamplification of antigen-specific T cell responses, has been shown to bea promising approach in human cancer therapeutics. Non-limiting examplesof immune checkpoints (ligands and receptors), some of which areselectively upregulated in various types of tumor cells, that arecandidates for blockade include PD1 (programmed cell death protein 1);PDL1 (PD1 ligand); BTLA (B and T lymphocyte attenuator); CTLA4(cytotoxic T-lymphocyte associated antigen 4); TIM3 (T-cell membraneprotein 3); LAG3 (lymphocyte activation gene 3); A2aR (adenosine A2areceptor A2aR); and Killer Inhibitory Receptors. Non-limiting examplesof immune checkpoint inhibitors include ipulimumab, nivolumab andlambrolizumab.

In other embodiments, there are provided methods for treating cancer ina subject, comprising administering to the subject a therapeuticallyeffective amount of at least one CD73 inhibitor compound or compositionthereof and at least one chemotherapeutic agent, such agents including,but not limited to alkylating agents (e.g., nitrogen mustards such aschlorambucil, cyclophosphamide, isofamide, mechlorethamine, melphalan,and uracil mustard; aziridines such as thiotepa; methanesulphonateesters such as busulfan; nucleoside analogs (e.g., gemcitabine); nitrosoureas such as carmustine, lomustine, and streptozocin; topoisomerase 1inhibitors (e.g., irinotecan); platinum complexes such as cisplatin andcarboplatin; bioreductive alkylators such as mitomycin, procarbazine,dacarbazine and altretamine); DNA strand-breakage agents (e.g.,bleomycin); topoisomerase II inhibitors (e.g., amsacrine, dactinomycin,daunorubicin, idarubicin, mitoxantrone, doxorubicin, etoposide, andteniposide); DNA minor groove binding agents (e.g., plicamydin);antimetabolites (e.g., folate antagonists such as methotrexate andtrimetrexate; pyrimidine antagonists such as fluorouracil,fluorodeoxyuridine, CB3717, azacitidine, cytarabine, and floxuridine;purine antagonists such as mercaptopurine, 6-thioguanine, fludarabine,pentostatin; asparginase; and ribonucleotide reductase inhibitors suchas hydroxyurea); tubulin interactive agents (e.g., vincristine,estramustine, vinblastine, docetaxol, epothilone derivatives, andpaclitaxel); hormonal agents (e.g., estrogens; conjugated estrogens;ethinyl estradiol; diethylstilbesterol; chlortrianisen; idenestrol;progestins such as hydroxyprogesterone caproate, medroxyprogesterone,and megestrol; and androgens such as testosterone, testosteronepropionate, fluoxymesterone, and methyltestosterone); adrenalcorticosteroids (e.g., prednisone, dexamethasone, methylprednisolone,and prednisolone); leutinizing hormone releasing agents orgonadotropin-releasing hormone antagonists (e.g., leuprolide acetate andgoserelin acetate); and antihormonal antigens (e.g., tamoxifen,antiandrogen agents such as flutamide; and antiadrenal agents such asmitotane and aminoglutethimide). There is also provided the use of theCD73 inhibitors in combination with other agents known in the art (e.g.,arsenic trioxide) and other chemotherapeutic agents that may bedeveloped in the future.

In some embodiments drawn to methods of treating cancer, theadministration of a therapeutically effective amount of a CD73 inhibitorin combination with at least one chemotherapeutic agent results in acancer survival rate greater than the cancer survival rate observed byadministering either agent alone. In further embodiments drawn tomethods of treating cancer, the administration of a therapeuticallyeffective amount of a CD73 inhibitor in combination with at least onechemotherapeutic agent results in a reduction of tumor size or a slowingof tumor growth greater than reduction of the tumor size or slowing oftumor growth observed by administration of either agent alone.

In further embodiments, there are provided methods for treating orpreventing cancer in a subject, comprising administering to the subjecta therapeutically effective amount of at least one CD73 inhibitorcompound or composition and at least one signal transduction inhibitor(STI). In a particular embodiment, the at least one STI is selected fromthe group consisting of bcr/abl kinase inhibitors, epidermal growthfactor (EGF) receptor inhibitors, her-2/neu receptor inhibitors, andfarnesyl transferase inhibitors (FTIs).

In other embodiments, there are provided methods of augmenting therejection of tumor cells in a subject comprising administering an CD73inhibitor compound or composition in conjunction with at least onechemotherapeutic agent and/or radiation therapy, wherein the resultingrejection of tumor cells is greater than that obtained by administeringeither the CD73 inhibitor, the chemotherapeutic agent or the radiationtherapy alone.

In further embodiments, there are provided methods for treating cancerin a subject, comprising administering to the subject a therapeuticallyeffective amount of at least one CD73 inhibitor and at least oneimmunomodulator other than a CD73 inhibitor. It should be understoodthat, as used herein, a “CD73 inhibitor” refers to compounds providedherein, e.g., a compound of any one of Formulae I-IX, a compound ofTable 1, or a pharmaceutically acceptable salt or ester thereof, and topharmaceutical compositions thereof.

In some embodiments, there are provided methods of treating orpreventing a CD73-associated disease, disorder or condition in a subjectin need thereof, comprising administering a therapeutically effectiveamount of at least one CD73 inhibitor or a pharmaceutical compositionthereof to the subject, such that the CD73-associated disease, disorderor condition is treated or prevented in the subject. In someembodiments, the compound is administered in an amount effective toreverse, slow or stop the progression of CD73-mediated immunosuppressionin the subject.

In some embodiments, the CD73-associated disease, disorder or conditionis cancer, such as, without limitation, a cancer of the prostate, colon,rectum, pancreas, cervix, stomach, endometrium, brain, liver, bladder,ovary, testis, head, neck, skin, mesothelial lining, white blood cell,esophagus, breast, muscle, connective tissue, lung, adrenal gland,thyroid, kidney, or bone. In some embodiments, the cancer isglioblastoma, mesothelioma, renal cell carcinoma, gastric carcinoma,sarcoma, choriocarcinoma, cutaneous basocellular carcinoma, ortesticular seminoma. In some embodiments, the cancer is melanoma, coloncancer, pancreatic cancer, breast cancer, prostate cancer, lung cancer,leukemia, a brain tumor, lymphoma, ovarian cancer, or Kaposi's sarcoma.

In some embodiments, the CD73-associated disease, disorder or conditionis an immune-related disease, disorder or condition selected from thegroup consisting of rheumatoid arthritis, kidney failure, lupus, asthma,psoriasis, colitis, pancreatitis, allergies, fibrosis, anemiafibromyalgia, Alzheimer's disease, congestive heart failure, stroke,aortic valve stenosis, arteriosclerosis, osteoporosis, Parkinson'sdisease, infections, Crohn's disease, ulcerative colitis, allergiccontact dermatitis, eczema, systemic sclerosis and multiple sclerosis.

In some embodiments, methods provided herein further compriseadministration of at least one additional therapeutic agent to thesubject. The at least one additional therapeutic agent may beadministered concomitantly or sequentially with the compound orcomposition described herein. In some embodiments, the at least oneadditional therapeutic agent is a chemotherapeutic agent, an immune-and/or inflammation-modulating agent, an anti-hypercholesterolemiaagent, or an anti-infective agent. In an embodiment, the at least oneadditional therapeutic agent is an immune checkpoint inhibitor, such as,without limitation, ipulimumab, nivolumab or lambrolizumab.

In some embodiments, there are provided methods for treating orpreventing an infective disorder (e.g., a viral infection) in a subject(e.g., a human) comprising administering to the subject atherapeutically effective amount of at least one CD73 inhibitor and atherapeutically effective amount of an anti-infective agent(s), such asone or more antimicrobial agents.

In additional embodiments, treatment of an infective disorder iseffected through the co-administration of a vaccine in combination withadministration of a therapeutically effective amount of a CD73 inhibitorprovided herein. In some embodiments, the vaccine is an anti-viralvaccine, including, for example, an anti-HIV vaccine. In otherembodiments, the vaccine is effective against tuberculosis or malaria.In still other embodiments, the vaccine is a tumor vaccine (e.g., avaccine effective against melanoma); the tumor vaccine can comprisegenetically modified tumor cells or a genetically modified cell line,including genetically modified tumor cells or a genetically modifiedcell line that has been transfected to express granulocyte-macrophagestimulating factor (GM-CSF). In particular embodiments, the vaccineincludes one or more immunogenic peptides and/or dendritic cells.

In certain embodiments drawn to treatment of an infection byadministering an CD73 inhibitor and at least one additional therapeuticagent, a symptom of infection observed after administering both the CD73inhibitor and the additional therapeutic agent is improved over the samesymptom of infection observed after administering either alone. In someembodiments, the symptom of infection observed can be reduction in viralload, increase in CD4+ T cell count, decrease in opportunisticinfections, increased survival time, eradication of chronic infection,or a combination thereof.

In some embodiments, there are provided methods of treating cancer in asubject, comprising administering to the subject an effective amount ofa compound or composition described herein and an immune checkpointinhibitor, such that cancer is treated in the subject. The compound orcomposition described herein and the immune checkpoint inhibitor may beadministered in combination or sequentially. The compound or compositionmay be administered after the immune checkpoint inhibitor or prior toadministration of the immune checkpoint inhibitor. In some embodiments,the compound or composition and/or the immune checkpoint inhibitor areadministered prior to, concurrent with, or subsequent to, otheranti-cancer treatment such as, without limitation, radiation treatment.In some embodiments, the immune checkpoint inhibitor is selected fromthe group consisting of ipulimumab, nivolumab and lambrolizumab.

In a fourth broad aspect, there are provided kits comprising thecompound or composition described herein. Kits may further comprise abuffer or excipient, and/or instructions for use. In some embodiments,kits further comprise at least one additional therapeutic agent, such aswithout limitation a chemotherapeutic agent, an immune- and/orinflammation-modulating agent, an anti-hypercholesterolemia agent, ananti-infective agent, or an immune checkpoint inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

For a better understanding of the invention and to show more clearly howit may be carried into effect, reference will now be made by way ofexample to the accompanying drawings, which illustrate aspects andfeatures according to embodiments of the present invention, and inwhich:

FIG. 1 is a graph showing the CD73 inhibition rate (% Inhibition vs.Log[Conc.]/nM) for compound 9; and

FIG. 2 is a graph showing the CD73 inhibition rate for compound 22.

DETAILED DESCRIPTION

The number of subjects diagnosed with cancer and the number of deathsattributable to cancer continue to rise. Traditional treatmentapproaches comprising chemotherapy and radiotherapy are generallydifficult for the subject to tolerate and become less effective ascancers (e.g., tumors) evolve to circumvent such treatments. Recentexperimental evidence indicates that CD73 inhibitors may represent animportant new treatment modality for cancer (e.g., breast cancer)treatment.

Promising data also support the role of inhibitors of CD73 function toinhibit the anti-inflammatory activity of CD73 and/or theimmunosuppressive activity of CD73, and thus CD73 inhibitors may beuseful to treat, for example, immunosuppressive diseases (e.g., HIV andAIDs). Inhibition of CD73 may also be an important treatment strategyfor subjects with neurological or neuropsychiatric diseases or disorderssuch as depression.

There are provided herein, inter alia, small molecule compounds havingCD73 inhibitory activity, as well as compositions thereof, and methodsof using the compounds and compositions for the treatment and preventionof the diseases, disorders and conditions described herein. Compoundsprovided herein are useful as inhibitors of CD73 and, therefore, usefulin the treatment of diseases, disorders, and conditions in which CD73activity plays a role. Additionally, the compounds provided herein maybe useful as inhibitors of adenosine receptors such as, for example, theA₂A receptor. Accordingly, the compounds provided herein are useful inthe treatment of diseases, disorders, and conditions associated withactivity of one or more adenosine receptors.

In an embodiment, there is provided herein a method of treating asubject (e.g., a human) with cancer or a disorder mediated by CD73comprising the step of administering to the subject a therapeuticallyeffective amount of an CD73 inhibitor provided herein, e.g., a compoundprovided herein or a pharmaceutically acceptable composition thereof.

It should be understood that a pharmaceutical composition comprises acompound disclosed herein (or a pharmaceutically acceptable salt orester thereof) and a pharmaceutically acceptable carrier, adjuvant, orvehicle. In certain embodiments, the amount of a compound in acomposition is such that it is effective as an inhibitor of CD73 in abiological sample (e.g., in an in vitro assay, in an in vivo model,etc.) or in a subject. In certain embodiments, the composition isformulated for administration to a subject in need of such composition.In some embodiments, the composition is an injectable formulation. Inother embodiments, the composition is formulated for oral administrationto a subject.

There is also provided a method of treating a subject (e.g., a human)with cancer or a disorder mediated by an adenosine receptor (e.g., A₂AR)comprising the step of administering to the subject a therapeuticallyeffective amount of an CD73 inhibitor provided herein, e.g., a compoundprovided herein or a pharmaceutically acceptable composition thereof. Incertain embodiments, the amount of a compound in a composition is suchthat it is effective as an inhibitor of an adenosine receptor (e.g.,A₂AR) in a biological sample (e.g., in an in vitro assay, in an in vivomodel, etc.) or in a subject. In certain embodiments, the composition isformulated for administration to a subject in need of such composition.In some embodiments, the composition is an injectable formulation. Inother embodiments, the composition is formulated for oral administrationto a subject. In some embodiments, the composition is in the form of ahard shell gelatin capsule, a soft shell gelatin capsule, a cachet, apill, a tablet, a lozenge, a powder, a granule, a pellet, a pastille, ora dragee. In some embodiments, the composition is in the form of asolution, an aqueous liquid suspension, a non-aqueous liquid suspension,an oil-in-water liquid emulsion, a water-in-oil liquid emulsion, anelixir, or a syrup. In some embodiments, the composition is entericcoated. In some embodiments, the composition is formulated forcontrolled release.

In further embodiments, there are provided methods for treating orpreventing cancer in a subject, comprising administering to the subjecta therapeutically effective amount of at least one CD73 inhibitor and atleast one signal transduction inhibitor (STI). In a particularembodiment, the at least one STI is selected from the group consistingof bcr/abl kinase inhibitors, epidermal growth factor (EGF) receptorinhibitors, her-2/neu receptor inhibitors, and farnesyl transferaseinhibitors (FTIs). There are also provided methods of augmenting therejection of tumor cells in a subject comprising administering an CD73inhibitor in conjunction with at least one chemotherapeutic agent and/orradiation therapy, wherein the resulting rejection of tumor cells isgreater than that obtained by administering either the CD73 inhibitor,the chemotherapeutic agent or the radiation therapy alone. In furtherembodiments, there are provided methods for treating cancer in asubject, comprising administering to the subject a therapeuticallyeffective amount of at least one CD73 inhibitor and at least oneimmunomodulator other than a CD73 inhibitor.

In other embodiments, there are provided methods for treating orpreventing an infective disorder (e.g., a viral infection) in a subject(e.g., a human) comprising administering to the subject atherapeutically effective amount of at least one CD73 inhibitor and atherapeutically effective amount of an anti-infective agent(s), such asone or more antimicrobial agents.

In additional embodiments, treatment of an infective disorder iseffected through the co-administration of a vaccine in combination withadministration of a therapeutically effective amount of an CD73inhibitor provided herein. In some embodiments, the vaccine is ananti-viral vaccine, including, for example, an anti-HIV vaccine. Inother embodiments, the vaccine is effective against tuberculosis ormalaria. In still other embodiments, the vaccine is a tumor vaccine(e.g., a vaccine effective against melanoma); the tumor vaccine cancomprise genetically modified tumor cells or a genetically modified cellline, including genetically modified tumor cells or a geneticallymodified cell line that has been transfected to expressgranulocyte-macrophage stimulating factor (GM-CSF). In particularembodiments, the vaccine includes one or more immunogenic peptidesand/or dendritic cells.

In certain embodiments drawn to treatment of an infection byadministering an CD73 inhibitor and at least one additional therapeuticagent, a symptom of infection observed after administering both the CD73inhibitor and the additional therapeutic agent is improved over the samesymptom of infection observed after administering either alone. In someembodiments, the symptom of infection observed can be reduction in viralload, increase in CD4+ T cell count, decrease in opportunisticinfections, increased survival time, eradication of chronic infection,or a combination thereof.

Definitions

In order to provide a clear and consistent understanding of the termsused in the present specification, a number of definitions are providedbelow. Moreover, unless defined otherwise, all technical and scientificterms as used herein have the same meaning as commonly understood to oneof ordinary skill in the art to which this invention pertains.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one”, butit is also consistent with the meaning of “one or more”, “at least one”,and “one or more than one”. Similarly, the word “another” may mean atleast a second or more.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “include” and “includes”) or “containing”(and any form of containing, such as “contain” and “contains”), areinclusive or open-ended and do not exclude additional, unrecitedelements or process steps.

The term “about” is used to indicate that a value includes an inherentvariation of error for the device or the method being employed todetermine the value.

The term “derivative” as used herein, is understood as being a substancesimilar in structure to another compound but differing in some slightstructural detail.

The present description refers to a number of chemical terms andabbreviations used by those skilled in the art. Nevertheless,definitions of selected terms are provided for clarity and consistency.

As used herein, the term “alkyl” refers to saturated hydrocarbons havingfrom one to thirty carbon atoms, including linear, branched, and cyclicalkyl groups. Examples of alkyl groups include, without limitation,methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl,decyl, isopropyl, tert-butyl, sec-butyl, isobutyl, cyclopropyl,cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like. The termalkyl includes both unsubstituted alkyl groups and substituted alkylgroups. The terms “C₁-C_(n)alkyl” and “C_(1-n) alkyl”, wherein n is aninteger from 2 to 30, are used interchangeably to refer to an alkylgroup having from 1 to the indicated “n” number of carbon atoms. Alkylresidues may be substituted or unsubstituted. In some embodiments, forexample, alkyl may be substituted by hydroxyl, amino, carboxyl,carboxylic ester, amide, carbamate, or aminoalkyl. In some particularembodiments, “alkyl” is modified by a range of the number of carbonatoms and thus the size of the alkyl group is defined specifically. Forexample, a C₁₁-C₃₀ alkyl specifies an alkyl group containing at least 11carbon atoms and not more than 30 carbon atoms.

As used herein, the term “acyclic” refers to an organic moiety without aring system. The term “aliphatic group” includes organic moietiescharacterized by straight or branched-chains, typically having between 1and 15 carbon atoms. Aliphatic groups include non-cyclic alkyl groups,alkenyl groups, and alkynyl groups.

As used herein, the term “alkenyl” refers to unsaturated hydrocarbonshaving from two to thirty carbon atoms, including linear, branched, andcyclic non aromatic alkenyl groups, and comprising between one to sixcarbon-carbon double bonds. Examples of alkenyl groups include, withoutlimitation, vinyl, allyl, 1-propen-2-yl, 1-buten-3-yl, 1-buten-4-yl,2-buten-4-yl, 1-penten-5-yl, 1,3-pentadien-5-yl, cyclopentenyl,cyclohexenyl, ethylcyclopentenyl, ethylcylohexenyl, and the like. Theterm alkenyl includes both unsubstituted alkenyl groups and substitutedalkenyl groups. The terms “C₂-C_(n)alkenyl” and “C_(2-n) alkenyl”,wherein n is an integer from 3 to 30, are used interchangeably to referto an alkenyl group having from 2 to the indicated “n” number of carbonatoms. In some particular embodiments, “alkenyl” is modified by a rangeof the number of carbon atoms and thus the size of the alkenyl group isdefined specifically. For example, a C₁₁-C₃₀ alkenyl specifies analkenyl group containing at least 11 carbon atoms and not more than 30carbon atoms.

As used herein, the term “alkynyl” refers to unsaturated hydrocarbonshaving from two to thirty carbon atoms, including linear, branched, andcyclic non aromatic alkynyl groups, and comprising between one to sixcarbon-carbon triple bonds. Examples of alkynyl groups include, withoutlimitation, ethynyl, 1-propyn-3-yl, 1-butyn-4-yl, 2-butyn-4-yl,1-pentyn-5-yl, 1,3-pentadiyn-5-yl, and the like. The term alkynylincludes both unsubstituted alkynyl groups and substituted alkynylgroups. The terms “C₂-C_(n)alkynyl” and “C_(2-n) alkynyl”, wherein n isan integer from 3 to 30, are used interchangeably to refer to an alkynylgroup having from 2 to the indicated “n” number of carbon atoms. In someparticular embodiments, “alkynyl” is modified by a range of the numberof carbon atoms and thus the size of the alkynyl group is definedspecifically. For example, a C₁₁-C₃₀ alkynyl specifies an alkynyl groupcontaining at least 11 carbon atoms and not more than 30 carbon atoms.

Unless the number of carbons is otherwise specified, “lower” as in“lower aliphatic,” “lower alkyl,” “lower alkenyl,” and “lower alkylnyl”,as used herein means that the moiety has at least one (two for alkenyland alkynyl) and equal to or less than 6 carbon atoms.

The terms “cycloalkyl”, “alicyclic”, “carbocyclic” and equivalentexpressions refer to a group comprising a saturated or partiallyunsaturated carbocyclic ring in a single, spiro (sharing one atom), orfused (sharing at least one bond) carbocyclic ring system having fromthree to fifteen ring members. Examples of cycloalkyl groups include,without limitation, cyclopropyl, cyclobutyl, cyclopentyl,cyclopenten-1-yl, cyclopenten-2-yl, cyclopenten-3-yl, cyclohexyl,cyclohexen-1-yl, cyclohexen-2-yl, cyclohexen-3-yl, cycloheptyl,bicyclo[4,3,0]nonanyl, norbornyl, and the like. The term cycloalkylincludes both unsubstituted cycloalkyl groups and substituted cycloalkylgroups. The terms “C₃-C_(n)cycloalkyl” and “C_(3-n) cycloalkyl”, whereinn is an integer from 4 to 15, are used interchangeably to refer to acycloalkyl group having from 3 to the indicated “n” number of carbonatoms in the ring structure. Unless the number of carbons is otherwisespecified, “lower cycloalkyl” groups as herein used, have at least 3 andequal to or less than 8 carbon atoms in their ring structure.

Cycloalkyl residues can be saturated or contain one or more double bondswithin the ring system. In particular they can be saturated or containone double bond within the ring system. In unsaturated cycloalkylresidues the double bonds can be present in any suitable positions.Monocycloalkyl residues are, for example, cyclopropyl, cyclobutyl,cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl,cycloheptenyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl,cyclododecyl or cyclotetradecyl, which can also be substituted, forexample by C₁₋₄ alkyl. Examples of substituted cycloalkyl residues are4-methylcyclohexyl and 2,3-dimethylcyclopentyl. Examples of parentstructures of bicyclic ring systems are norbornane,bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane and bicyclo[3.2.1]octane.

The term “heterocycloalkyl” and equivalent expressions refers to a groupcomprising a saturated or partially unsaturated carbocyclic ring in asingle, spiro (sharing one atom), or fused (sharing at least one bond)carbocyclic ring system having from three to fifteen ring members,including one to six heteroatoms (e.g., N, O, S, P) or groups containingsuch heteroatoms (e.g., NH, NRx (Rx is alkyl, acyl, aryl, heteroaryl orcycloalkyl), PO₂, SO, SO₂, and the like). Heterocycloalkyl groups may beC-attached or heteroatom-attached (e.g., via a nitrogen atom) where suchis possible. Examples of heterocycloalkyl groups include, withoutlimitation, pyrrolidino, tetrahydrofuranyl, tetrahydrodithienyl,tetrahydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino,thiomorpholino, thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl,homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl,thiazepinyl, 1,2,3,6-tetrahydropyridinyl, 2-pyrrolinyl, 3-pyrrolinyl,indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl,pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl,dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl,3-azabicyclo[3,1,0]hexanyl, 3-azabicyclo[4,1,0]heptanyl, 3H-indolyl,quinolizinyl, and sugars, and the like. The term heterocycloalkylincludes both unsubstituted heterocycloalkyl groups and substitutedheterocycloalkyl groups. The terms “C₃-C_(n)heterocycloalkyl” and“C_(3-n) heterocycloalkyl”, wherein n is an integer from 4 to 15, areused interchangeably to refer to a heterocycloalkyl group having from 3to the indicated “n” number of atoms in the ring structure, including atleast one hetero group or atom as defined above. Unless the number ofcarbons is otherwise specified, “lower heterocycloalkyl” groups asherein used, have at least 3 and equal to or less than 8 carbon atoms intheir ring structure.

The terms “aryl” and “aryl ring” refer to aromatic groups having “4n+2”(pi) electrons, wherein n is an integer from 1 to 7, in a conjugatedmonocyclic or polycyclic system (fused or not) and having six tofourteen ring atoms. A polycyclic ring system includes at least onearomatic ring. Aryl may be directly attached, or connected via a C₁-C₆alkyl group (also referred to as arylalkyl or aralkyl). Examples of arylgroups include, without limitation, phenyl, benzyl, phenetyl,1-phenylethyl, tolyl, naphthyl, biphenyl, terphenyl, indenyl,benzocyclooctenyl, benzocycloheptenyl, azulenyl, acenaphthylenyl,fluorenyl, phenanthernyl, anthracenyl, and the like. The term arylincludes both unsubstituted aryl groups and substituted aryl groups. Theterms “C₆-C_(n)aryl” and “C_(6-n) aryl”, wherein n is an integer from 6to 30, are used interchangeably to refer to an aryl group having from 6to the indicated “n” number of atoms in the ring structure, including atleast one hetero group or atom as defined above.

The terms “heteroaryl” and “heteroaryl ring” refer to an aromatic grouphaving “4n+2” (pi) electrons, wherein n is an integer from 1 to 7, in aconjugated monocyclic or polycyclic system (fused or not) and havingfive to fourteen ring members, including one to six heteroatoms (e.g. N,O, S) or groups containing such heteroatoms (e.g. NH, NRx (Rx is alkyl,acyl, aryl, heteroaryl or cycloalkyl), SO, and the like). A polycyclicring system includes at least one heteroaromatic ring. Heteroaryls maybe directly attached, or connected via a C₁-C₃alkyl group (also referredto as heteroarylalkyl or heteroaralkyl). Heteroaryl groups may beC-attached or heteroatom-attached (e.g., via a nitrogen atom), wheresuch is possible. Examples of heteroaryl groups include, withoutlimitation, pyridyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl,tetrazolyl, furyl, thienyl; isooxazolyl, thiazolyl, oxazolyl,isothiazolyl, pyrrollyl, quinolinyl, isoquinolinyl, indolyl, isoindolyl,chromenyl, isochromenyl, benzimidazolyl, benzofuranyl, cinnolinyl,indazolyl, indolizinyl, phthalazinyl, pyridazinyl, pyrazinyl, triazinyl,isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl,benzofurazanyl, benzothiophenyl, benzothienyl, benzothiazolyl,benzoxazolyl, quinazolinyl, quinolizinyl, quinolonyl, isoquinolonyl,quinoxalinyl, naphthyridinyl, furopyridinyl, carbazolyl,phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, phenazinyl,phenothiazinyl, phenoxazinyl, dibenzofuranyl, and the like. The termheteroaryl includes both unsubstituted heteroaryl groups and substitutedheteroaryl groups. The terms “C₅-C_(n)heteroaryl” and “C_(5-n)heteroaryl”, wherein n is an integer from 6 to 29, are usedinterchangeably to refer to a heteroaryl group having from 5 to theindicated “n” number of atoms in the ring structure, including at leastone hetero group or atom as defined above.

The terms “heterocycle” or “heterocyclic” include heterocycloalkyl andheteroaryl groups. Examples of heterocycles include, without limitation,acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl,benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl,benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl,carbazolyl, 4αH-carbazolyl, carbolinyl, chromanyl, chromenyl,cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isobenzofuranyl, isochromanyl,isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl,isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl,octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxazolidinyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl,piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole,pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl,pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl,quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl,1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl,xanthenyl, and the like. The term heterocycle includes bothunsubstituted heterocyclic groups and substituted heterocyclic groups.

The term “amine” or “amino,” as used herein, refers to an unsubstitutedor substituted moiety of the formula —NRaRb, in which Ra and Rb are eachindependently hydrogen, alkyl, aryl, or heterocyclyl, or Ra and Rb,taken together with the nitrogen atom to which they are attached, form aheterocyclic ring. The term amino includes compounds or moieties inwhich a nitrogen atom is covalently bonded to at least one carbon orheteroatom. Thus, the terms “alkylamino” and “dialkylamino” as usedherein mean an amine group having respectively one and at least twoC₁-C₆alkyl groups attached thereto. The terms “arylamino” and“diarylamino” include groups wherein the nitrogen is bound to at leastone or two aryl groups, respectively. The terms “amide” or“aminocarbonyl” include compounds or moieties which contain a nitrogenatom which is bound to the carbon of a carbonyl or a thiocarbonyl group.The term “acylamino” refers to an amino group directly attached to anacyl group as defined herein.

The term “bicycle” or “bicyclic” refers to a ring system with two ringsthat has two ring carbon atoms in common, and which can be located atany position along either ring, generally referring to bicyclichydrocarbon radical, bicyclic aromatic carbon atom ring structureradical, and a saturated or partially unsaturated bicyclic carbon atomring structure radical in which one or more carbon atom ring membershave been replaced, where allowed by structural stability, with aheteroatom, such as an O, S or N atom. The bicyclic system can be afused-ring system, such as bicyclo[4.4.0]decane or naphthalene, or abridged-ring system, such as bicyclo[2.2.2]octane.

The term “tricycle” or “tricyclic” refers to a ring system with threerings that has three ring carbon atoms in common, and which can belocated at any position along each ring; generally referring totricyclic hydrocarbon radical, tricyclic aromatic carbon atom ringstructure radical, and a saturated or partially unsaturated tricycliccarbon atom ring structure radical in which one or more carbon atom ringmembers have been replaced, where allowed by structural stability, witha heteroatom, such as an O, S or N atom. A tricyclic system can havethree rings arranged as a fused ring, such as anthracene ortetradecahydroanthracene, or a bridged ring, such as in adamantine ortricycle[3.3.1.1]decane.

The term “multi-cycle”, “multicycle”, “multi-cyclic”, or “multi-cyclic”means a ring system with more than three rings having more than threering carbon atoms in common, and which can be located at any positionalong either ring. The term generally refers to a multicyclichydrocarbon radical, a multicyclic aromatic carbon atom ring structureradical, and a saturated or partially unsaturated multicyclic carbonatom ring structure radical in which one or more carbon atom ringmembers have been replaced, where allowed by structural stability, witha heteroatom, such as an O, S or N atom.

The term “fused ring” or “fused” refers to a polycyclic ring system thatcontains fused rings. Typically, a fused ring system contains 2 or 3rings and/or up to 18 ring atoms. As defined above, cycloalkyl radicals,aryl radicals and heterocyclyl radicals may form fused ring systems.Thus, a fused ring system may be aromatic, partially aromatic or notaromatic and may contain heteroatoms. A spiro ring system is not afused-polycyclic by this definition, but fused polycyclic ring systemsof the invention may themselves have spiro rings attached thereto via asingle ring atom of the system. Examples of fused ring systems include,but are not limited to, naphthyl (e.g. 2-naphthyl), indenyl, fenanthryl,anthracyl, pyrenyl, benzimidazole, benzothiazole, etc.

The term “spiral ring” or “spiral” refers to an organic compound, thatpresents a twisted structure of two or more rings (a ring system), inwhich 2 or 3 rings are linked together by one common atom. Spirocompounds may be fully carbocyclic (all carbon), such as withoutlimitation spiro[5.5]undecane or heterocyclic (having one or morenon-carbon atom), including but not limited to carbocyclic spirocompounds, heterocyclic spiro compounds and polyspiro compounds.

The term “bridged ring” or “bridged” refers to a carbocyclic orheterocyclic moiety where two or more atoms are shared between two ormore ring structures, where any such shared atom is C, N, S, or otherheteroatom arranged in a chemically reasonable substitution pattern.Alternatively, a “bridged” compound also refers to a carbocyclic orheterocyclic ring structure where one atom at any position of a primaryring is bonded to a second atom on the primary ring through either achemical bond or atom (s) other than a bond which does (do) not comprisea part of the primary ring structure. The first and second atom may ormay not be adjacent to one another in the primary ring. Illustratedbelow are specific non-limiting examples of bridged ring structurescontemplated herein. Other carbocyclic or heterocyclic bridged ringstructures are also contemplated, including bridged rings wherein thebridging atoms are C or heteroatom (s) arranged in chemically reasonablesubstitution patterns, as are known in the art.

The term “nitro” means —NO₂; the terms “halo” and “halogen” refer tobromine, chlorine, fluorine or iodine substituents; the terms “thiol”,“thio”, and “mercapto” mean —SH; and the terms “hydroxyl” and “hydroxy”mean —OH. The term “alkylthio” refers to an alkyl group, having asulfhydryl group attached thereto. Suitable alkylthio groups includegroups having 1 to about 12 carbon atoms, preferably from 1 to about 6carbon atoms. The term “alkylcarboxyl” as used herein means an alkylgroup having a carboxyl group attached thereto.

The terms “alkoxy” and “lower alkoxy” as used herein mean an alkyl grouphaving an oxygen atom attached thereto. Representative alkoxy groupsinclude groups having 1 to about 6 carbon atoms, e.g., methoxy, ethoxy,propoxy, tert-butoxy and the like. Examples of alkoxy groups includemethoxy, ethoxy, isopropyloxy, propoxy, butoxy, pentoxy, fluoromethoxy,difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy,trichloromethoxy groups, and the like. The term “alkoxy” includes bothunsubstituted or substituted alkoxy groups, etc., as well asperhalogenated alkyloxy groups.

The terms “carbonyl” and “carboxy” include compounds and moieties whichcontain a carbon connected with a double bond to an oxygen atom.Examples of moieties which contain a carbonyl include aldehydes,ketones, carboxylic acids, amides, esters, anhydrides, etc.

The term “acyl” refers to a carbonyl group that is attached through itscarbon atom to a hydrogen (i.e., formyl), an aliphatic group (e.g.,C₁-C₂₉ alkyl, C₁-C₂₉ alkenyl, C₁-C₂₉ alkynyl, e.g., acetyl), acycloalkyl group (C₃-C₈cycloalkyl), a heterocyclic group(C₃-C₈heterocycloalkyl and C₅-C₆heteroaryl), an aromatic group (C₆aryl,e.g., benzoyl), and the like. Acyl groups may be unsubstituted orsubstituted acyl groups (e.g., salicyloyl).

It should be understood that “substitution” or “substituted with”includes the implicit proviso that such substitution is in accordancewith the permitted valence of the substituted atom and the substituent,and that the substitution results in a stable compound, i.e., a compoundwhich does not spontaneously undergo transformation such as byrearrangement, cyclization, elimination, etc. As used herein, the term“substituted” is meant to include all permissible substituents oforganic compounds. In a broad aspect, the permissible substituentsinclude acyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic substituents of organiccompounds. The permissible substituents can be one or more. The term“substituted”, when used in association with any of the foregoing groupsrefers to a group substituted at one or more position with substituentssuch as acyl, amino (including simple amino, mono and dialkylamino, monoand diarylamino, and alkylarylamino), acylamino (including carbamoyl,and ureido), alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,alkoxycarbonyl, carboxy, carboxylate, aminocarbonyl, mono anddialkylaminocarbonyl, cyano, azido, halogen, hydroxyl, nitro,trifluoromethyl, thio, alkylthio, arylthio, alkylthiocarbonyl,thiocarboxylate, lower alkyl, lower alkenyl, lower alkynyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, lower alkoxy, aryloxy,aryloxycarbonyloxy, benzyloxy, benzyl, sulfinyl, alkylsulfinyl,sulfonyl, sulfate, sulfonate, sulfonamide, phosphate, phosphonato,phosphinato, oxo, guanidine, imino, formyl and the like. Any of theabove substituents can be further substituted if permissible, e.g., ifthe group contains an alkyl group, an aryl group, or other.

The term “solvate” refers to a physical association of a compound withone or more solvent molecules, whether organic or inorganic. Thisphysical association includes hydrogen bonding. In certain instances, asolvate will be capable of isolation, for example when one or moresolvent molecules are incorporated in the crystal lattice of acrystalline solid. “Solvate” encompasses both solution-phase andisolable solvates. Exemplary solvates include, without limitation,hydrates, ethanolates, methanolates, hemiethanolates, and the like.

A “pharmaceutically acceptable salt” of a compound means a salt of acompound that is pharmaceutically acceptable. Desirable are salts of acompound that retain or improve the biological effectiveness andproperties of the free acids and bases of the parent compound as definedherein or that take advantage of an intrinsically basic, acidic orcharged functionality on the molecule and that are not biologically orotherwise undesirable. Examples of pharmaceutically acceptable salts arealso described, for example, in Berge et al., “Pharmaceutical Salts”, J.Pharm. Sci. 66, 1-19 (1977). Non-limiting examples of such saltsinclude:

(1) acid addition salts, formed on a basic or positively chargedfunctionality, by the addition of inorganic acids such as hydrochloricacid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid,nitric acid, phosphoric acid, carbonate forming agents, and the like; orformed with organic acids such as acetic acid, propionic acid, lacticacid, oxalic, glycolic acid, pivalic acid, t-butylacetic acid,β-hydroxybutyric acid, valeric acid, hexanoic acid,cyclopentanepropionic acid, pyruvic acid, malonic acid, succinic acid,malic acid, maleic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelicacid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonicacid, 2-hydroxyethanesulfonic acid, cyclohexylaminosulfonic acid,benzenesulfonic acid, sulfanilic acid, 4-chlorobenzenesulfonic acid,2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonicacid, 3-phenyl propionic acid, lauryl sulphonic acid, lauryl sulfuricacid, oleic acid, palmitic acid, stearic acid, lauric acid, embonic(pamoic) acid, palmoic acid, pantothenic acid, lactobionic acid, alginicacid, galactaric acid, galacturonic acid, gluconic acid, glucoheptonicacid, glutamic acid, naphthoic acid, hydroxynapthoic acid, salicylicacid, ascorbic acid, stearic acid, muconic acid, and the like;

(2) base addition salts, formed when an acidic proton present in theparent compound either is replaced by a metal ion, including, an alkalimetal ion (e.g., lithium, sodium, potassium), an alkaline earth ion(e.g., magnesium, calcium, barium), or other metal ions such asaluminum, zinc, iron and the like; or coordinates with an organic basesuch as ammonia, ethylamine, diethylamine, ethylenediamine,N,N′-dibenzylethylenediamine, ethanolamine, diethanolamine,triethanolamine, tromethamine, N-methylglucamine, piperazine,chloroprocain, procain, choline, lysine and the like.

Pharmaceutically acceptable salts may be synthesized from a parentcompound that contains a basic or acidic moiety, by conventionalchemical methods. Generally, such salts are prepared by reacting thefree acid or base forms of compounds with a stoichiometric amount of theappropriate base or acid in water or in an organic solvent, or in amixture of the two. Salts may be prepared in situ, during the finalisolation or purification of a compound or by separately reacting acompound in its free acid or base form with the desired correspondingbase or acid, and isolating the salt thus formed. The term“pharmaceutically acceptable salts” also include zwitterionic compoundscontaining a cationic group covalently bonded to an anionic group, asthey are “internal salts”. It should be understood that all acid, salt,base, and other ionic and non-ionic forms of compounds described hereinare intended to be encompassed. For example, if a compound is shown asan acid herein, the salt forms of the compound are also encompassed.Likewise, if a compound is shown as a salt, the acid and/or basic formsare also encompassed.

Compounds provided herein may contain unnatural proportions of atomicisotopes at one or more of the atoms that constitute such compounds.Unnatural proportions of an isotope may be defined as ranging from theamount found in nature to an amount consisting of 100% of the atom inquestion. For example, compounds may incorporate radioactive isotopes,such as for example tritium (³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C),or non-radioactive isotopes, such as deuterium (²H) or carbon-13 (¹³C).Such isotopic variations can provide additional utilities to thosedescribed elsewhere within this application. For instance, isotopicvariants of the compounds of the invention may find additional utility,including but not limited to, as diagnostic and/or imaging reagents, oras cytotoxic/radiotoxic therapeutic agents. Additionally, isotopicvariants can have altered pharmacokinetic and pharmacodynamiccharacteristics which can contribute to enhanced safety, tolerability orefficacy during treatment. All isotopic variations of compounds providedherein, whether radioactive or not, are intended to be encompassedherein.

Isotopic enrichment is a process by which the relative abundance of theisotopes of a given element are altered, thus producing a form of theelement that has been enriched (i.e., increased) in one particularisotope and reduced or depleted in its other isotopic forms. As usedherein, an “isotope-enriched” compound or derivative refers to acompound in which one or more specific isotopic form has been increased,i.e., one or more of the elements has been enriched (i.e., increased) inone or more particular isotope. Generally, in an isotope-enrichedcompound or derivative, a specific isotopic form of an element at aspecific position of the compound is increased. It should be understoodhowever that isotopic forms of two or more elements in the compound maybe increased. Further, an isotope-enriched compound may be a mixture ofisotope-enriched forms that are enriched for more than one particularisotope, more than one element, or both. As used herein, an“isotope-enriched” compound or derivative possesses a level of anisotopic form that is higher than the natural abundance of that form.The level of isotope-enrichment will vary depending on the naturalabundance of a specific isotopic form. In some embodiments, the level ofisotope-enrichment for a compound, or for an element in a compound, maybe from about 2 to about 100 molar percent (%), e.g., about 2%, about5%, about 17%, about 30%, about 51%, about 83%, about 90%, about 95%,about 96%, about 97%, about 98%, greater than about 98%, about 99%, or100%.

As used herein, an “element of natural abundance” and an “atom ofnatural abundance” refers to the element or atom respectively having theatomic mass most abundantly found in nature. For example, hydrogen ofnatural abundance is ¹H (protium); nitrogen of natural abundance is ¹⁴N;oxygen of natural abundance is ¹⁶O; carbon of natural abundance is ¹²C;and so on. A “non-isotope enriched” compound is a compound in which allthe atoms or elements in the compound are isotopes of natural abundance,i.e., all the atoms or elements have the atomic mass most abundantlyfound in nature.

The terms “patient” and “subject” are used interchangeably herein torefer to a human or a non-human animal (e.g., a mammal).

The terms “administration”, “administer” and the like, as they apply to,for example, a subject, cell, tissue, organ, or biological fluid, referto contact of, for example, an inhibitor of CD73, a pharmaceuticalcomposition comprising same, or a diagnostic agent to the subject, cell,tissue, organ, or biological fluid. In the context of a cell,administration includes contact (e.g., in vitro or ex vivo) of a reagentto the cell, as well as contact of a reagent to a fluid, where the fluidis in contact with the cell.

The terms “treat”, “treating”, treatment” and the like refer to a courseof action (such as administering an inhibitor of CD73 or apharmaceutical composition comprising same) initiated after a disease,disorder or condition, or a symptom thereof, has been diagnosed,observed, and the like, so as to eliminate, reduce, suppress, mitigate,or ameliorate, either temporarily or permanently, at least one of theunderlying causes of a disease, disorder, or condition afflicting asubject, or at least one of the symptoms associated with a disease,disorder, condition afflicting a subject. Thus, treatment includesinhibiting (e.g., arresting the development or further development ofthe disease, disorder or condition or clinical symptoms associationtherewith) an active disease.

The term “in need of treatment” as used herein refers to a judgment madeby a physician or other caregiver that a subject requires or willbenefit from treatment. This judgment is made based on a variety offactors that are in the realm of the physician's or caregiver'sexpertise.

The terms “prevent”, “preventing”, “prevention” and the like refer to acourse of action (such as administering a CD73 inhibitor or apharmaceutical composition comprising same) initiated in a manner (e.g.,prior to the onset of a disease, disorder, condition or symptom thereof)so as to prevent, suppress, inhibit or reduce, either temporarily orpermanently, a subject's risk of developing a disease, disorder,condition or the like (as determined by, for example, the absence ofclinical symptoms) or delaying the onset thereof: generally in thecontext of a subject predisposed to having a particular disease,disorder or condition. In certain instances, the terms also refer toslowing the progression of the disease, disorder or condition orinhibiting progression thereof to a harmful or otherwise undesiredstate.

The term “in need of prevention” as used herein refers to a judgmentmade by a physician or other caregiver that a subject requires or willbenefit from preventative care. This judgment is made based on a varietyof factors that are in the realm of a physician's or caregiver'sexpertise.

The terms “therapeutically effective amount” and “effective amount” areused interchangeably herein to refer to the administration of an agentto a subject, either alone or as part of a pharmaceutical compositionand either in a single dose or as part of a series of doses, in anamount capable of having any detectable, positive effect on any symptom,aspect, or characteristic of a disease, disorder or condition whenadministered to the subject. The therapeutically effective amount can beascertained by measuring relevant physiological effects, and it can beadjusted in connection with the dosing regimen and diagnostic analysisof the subject's condition, and the like. By way of example, measurementof the serum level of a CD73 inhibitor (or, e.g., a metabolite thereof)at a particular time post-administration may be indicative of whether atherapeutically effective amount has been used. In some embodiments, theterms “therapeutically effective amount” and “effective amount” refer tothe amount or dose of a therapeutic agent, such as a compound, uponsingle or multiple dose administration to a subject, which provides thedesired therapeutic, diagnostic, or prognostic effect in the subject. Aneffective amount can be readily determined by an attending physician ordiagnostician using known techniques and by observing results obtainedunder analogous circumstances. In determining the effective amount ordose of compound administered, a number of factors are consideredincluding, but not limited to: the size, age, and general health of thesubject; the specific disease involved; the degree of or involvement orthe severity of the disease or condition to be treated; the response ofthe individual subject; the particular compound administered; the modeof administration; the bioavailability characteristics of thepreparation administered; the dose regimen selected; the use ofconcomitant medication(s); and other relevant considerations.

The term “substantially pure” is used herein to indicate that acomponent makes up greater than about 50% of the total content of thecomposition, and typically greater than about 60% of the total content.More typically, “substantially pure” refers to compositions in which atleast 75′%, at least 85%), at least 90% or more of the total compositionis the component of interest. In some cases, the component of interestwill make up greater than about 90%), or greater than about 95%) of thetotal content of the composition.

As used herein, the terms “CD73-associated disease, disorder orcondition” and “disease, disorder or condition mediated by CD73” areused interchangeably to refer to any disease, disorder or condition forwhich treatment with a CD73 inhibitor may be beneficial. In general,CD73-associated or mediated diseases, disorders and conditions are thosein which CD73 activity plays a biological, mechanistic, or pathologicalrole. Such diseases, disorders and conditions may also be associatedwith activity of one or more adenosine receptors. Non-limiting examplesof CD73-associated diseases, disorders and conditions includeoncology-related disorders (cancers, tumors, etc.), immune-relateddisorders, disorders with an inflammatory component, microbial-relateddisorders, CNS-related and neurological disorders, and other disorders(such as, without limitation, cardiovascular, gastrointestinal,metabolic, hepatic, pulmonary, ophthalmologic, and renal disorders).

For example, a CD73 inhibitor may be used to prevent or treat aproliferative condition, cancer or tumor; to increase or enhance animmune response; to improve immunization, including increasing vaccineefficacy; and to increase inflammation. Immune deficiencies associatedwith immune deficiency diseases, immunosuppressive medical treatment,acute and/or chronic infection, and aging can be treated using CD73inhibitors disclosed herein. CD73 inhibitors can also be used tostimulate the immune system of patients suffering fromiatrogenically-induced immune suppression, including those who haveundergone bone marrow transplants, chemotherapy, or radiotherapy. Inother embodiments, a CD73 inhibitor may be used to treat or prevent anyviral, bacterial, fungal, parasitic or other infective disease, disorderor condition, including without limitation HIV and AIDS.

In some embodiments, a CD73 inhibitor may be used to prevent or treat animmune-related disease, disorder or condition selected from the groupconsisting of rheumatoid arthritis, kidney failure, lupus, asthma,psoriasis, colitis, pancreatitis, allergies, fibrosis, anemiafibromyalgia, Alzheimer's disease, congestive heart failure, stroke,aortic valve stenosis, arteriosclerosis, osteoporosis, Parkinson'sdisease, infections, Crohn's disease, ulcerative colitis, allergiccontact dermatitis, eczema, systemic sclerosis and multiple sclerosis.

Pharmaceutical compositions provided herein can be formulated to becompatible with the intended method or route of administration;exemplary routes of administration are set forth herein. Furthermore,the pharmaceutical compositions may be used in combination with othertherapeutically active agents or compounds as described herein in orderto treat or prevent the CD73-associated diseases, disorders andconditions as contemplated herein.

Pharmaceutical compositions containing the active ingredient (e.g., aCD73 inhibitor) may be in a form suitable for oral use, for example, astablets, capsules, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules, emulsions, hard or sofi: capsules, orsyrups, solutions, microbeads or elixirs. Pharmaceutical compositionsintended for oral use may be prepared according to any method known inthe art for the manufacture of pharmaceutical compositions, and suchcompositions may contain one or more agents such as, for example,sweetening agents, flavoring agents, coloring agents and preservingagents in order to provide pharmaceutically acceptable preparations.Tablets, capsules and the like generally contain the active ingredientin admixture with non-toxic pharmaceutically acceptable carriers orexcipients which are suitable for the manufacture of tablets. Thesecarriers or excipients may be, for example, diluents, such as calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate; granulating and disintegrating agents, for example, cornstarch, or alginic acid; binding agents, for example starch, gelatin oracacia, and lubricating agents, for example magnesium stearate, stearicacid or talc.

Tablets, capsules and the like suitable for oral administration may beuncoated or coated using known techniques to delay disintegration andabsorption in the gastrointestinal tract and thereby provide a sustainedaction. For example, a time-delay material such as glyceryl monostearateor glyceryl distearate may be employed. They may also be coated bytechniques known in the art to form osmotic therapeutic tablets forcontrolled release. Additional agents include biodegradable orbiocompatible particles or a polymeric substance such as polyesters,polyamine acids, hydrogel, polyvinyl pyrrolidone, polyanhydrides,polyglycolic acid, ethylenevinylacetate, methycellulose,carboxymethylcellulose, protamine sulfate, or lactide/glycolidecopolymers, polylactide/glycolide copolymers, or ethylenevinylacetatecopolymers in order to control delivery of an administered composition.For example, the oral agent can be entrapped in microcapsules preparedby coacervation techniques or by interfacial polymerization, by the useof hydroxymethylcellulose or gelatin-microcapsules or poly(methylmethacrolate) microcapsules, respectively, or in a colloid drugdelivery system. Colloidal dispersion systems include macromoleculecomplexes, nano-capsules, microspheres, microbeads, and lipid-basedsystems, including oil-in-water emulsions, micelles, mixed micelles, andliposomes. Methods for the preparation of the above-mentionedformulations will be apparent to those skilled in the art.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate, kaolin ormicrocrystalline cellulose, or as soft gelatin capsules wherein theactive ingredient is mixed with water or an oil medium, for examplepeanut oil, liquid paraffin, or olive oil. Aqueous suspensions containthe active materials in admixture with excipients suitable for themanufacture thereof. Such excipients can be suspending agents, forexample sodium carboxymethylcellulose, methykellulose,hydroxy-propylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone,gum tragacanth and gum acacia; dispersing or wetting agents, for examplea naturally-occurring phosphatide (e.g., lecithin), or condensationproducts of an alkylene oxide with fatty acids (e.g., polyoxy-ethylenestearate), or condensation products of ethylene oxide with long chainaliphatic alcohols (e.g., for heptadecaethyleneoxycetanol), orcondensation products of ethylene oxide with partial esters derived fromfatty acids and a hexitol (e.g., polyoxyethylene sorbitol monooleate),or condensation products of ethylene oxide with partial esters derivedfrom fatty acids and hexitol anhydrides (e.g., polyethylene sorbitanmonooleate). The aqueous suspensions may also contain one or morepreservatives.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are known in the art.

Pharmaceutical compositions of the present invention may also be in theform of oil-in-water emulsions. The oily phase may be a vegetable oil,for example olive oil or arachis oil, or a mineral oil, for example,liquid paraffin, or mixtures of these. Suitable emulsifying agents maybe naturally occurring gums, for example, gum acacia or gum tragacanth;naturally occurring phosphatides, for example, soy bean, lecithin, andesters or partial esters derived from fatty acids; hexitol anhydrides,for example, sorbitan monooleate; and condensation products of partialesters with ethylene oxide, for example, polyoxyethylene sorbitanmonooleate.

Pharmaceutical compositions typically comprise a therapeuticallyeffective amount of a CD84 inhibitor compound provided herein and one ormore pharmaceutically and physiologically acceptable formulation agents.Suitable pharmaceutically acceptable or physiologically acceptablediluents, carriers or excipients include, but are not limited to,antioxidants (e.g., ascorbic acid and sodium bi sulfate), preservatives(e.g., benzyl alcohol, methyl parabens, ethyl or n-propyl,p-hydroxybenzoate), emulsifying agents, suspending agents, dispersingagents, solvents, fillers, bulking agents, detergents, buffers,vehicles, diluents, and/or adjuvants. For example, a suitable vehiclemay be physiological saline solution or citrate buffered saline,possibly supplemented with other materials common in pharmaceuticalcompositions for parenteral administration. Neutral buffered saline orsaline mixed with serum albumin are further exemplary vehicles. Thoseskilled in the art will readily recognize a variety of buffers that canbe used in the pharmaceutical compositions and dosage forms contemplatedherein. Typical buffers include, but are not limited to,pharmaceutically acceptable weak acids, weak bases, or mixtures thereof.As an example, the buffer components can be water soluble materials suchas phosphoric acid, tartaric acids, lactic acid, succinic acid, citricacid, acetic acid, ascorbic acid, aspartic acid, glutamic acid, andsalts thereof. Acceptable buffering agents include, for example, a Trisbuffer, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),2-(N-MoqJholino)ethanesulfonic acid (MES),2-(N-Morpholino)ethanesulfonic acid sodium salt (MES),3-(N-Morpholino)propanesulfonic acid (MOPS), andNtris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS). After apharmaceutical composition has been formulated, it may be stored insterile vials as a solution, suspension, gel, emulsion, solid, ordehydrated or lyophilized powder. Such formulations may be stored eitherin a ready-to-use form, a lyophilized form requiring reconstitutionprior to use, a liquid form requiring dilution prior to use, or otheracceptable form.

In some embodiments, the pharmaceutical composition is provided in asingle-use container (e.g., a single-use vial, ampoule, syringe, orautoinjector, whereas a multi-use container (e.g., a multi-use vial) isprovided in other embodiments.

Formulations can also include carriers to protect the compositionagainst rapid degradation or elimination from the body, such as acontrolled release formulation, including liposomes, hydrogels, andmicroencapsulated delivery systems. For example, a time delay materialsuch as glyceryl monostearate or glyceryl stearate alone, or incombination with a wax, may be employed. Any drug delivery apparatus maybe used to deliver a CD73 inhibitor, including implants (e.g.,implantable pumps) and catheter systems, slow injection pumps anddevices, all of which are well known to the skilled artisan.

Pharmaceutical compositions may also be in the form of a sterileinjectable aqueous or oleagenous suspension. This suspension may beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents mentioned herein. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butane diol. Acceptable diluents,solvents and dispersion media that may be employed include water,Ringer's solution, isotonic sodium chloride solution, Cremophor ELTM(BASF, Parsippany, N.J.) or phosphate buffered saline (PBS), ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol), and suitable mixtures thereof. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed, including synthetic mono-or diglycerides. Moreover, fatty acids such as oleic acid, find use inthe preparation of injectables. Prolonged absorption of particularinjectable formulations can be achieved by including an agent thatdelays absorption (e.g., aluminum monostearate or gelatin).

CD73 inhibitor compounds and compositions provided herein may beadministered to a subject in any appropriate manner known in the art.Suitable routes of administration include, without limitation: oral,parenteral (e.g., intramuscular, intravenous, subcutaneous (e.g.,injection or implant), intraperitoneal, intracisternal, intraarticular,intraperitoneal, intracerebral (intraparenchymal) andintracerebroventricular), nasal, vaginal, sublingual, intraocular,rectal, topical (e.g., transdermal), buccal and inhalation. Depotinjections, which are generally administered subcutaneously orintramuscularly, may also be utilized to release the CD73 inhibitorsdisclosed herein over a defined period of time. In certain embodiments,CD73 inhibitor compounds and compositions are administered orally to asubject in need thereof.

CD73 inhibitor compounds and compositions provided herein may beadministered to a subject in an amount that is dependent upon, forexample, the goal of administration (e.g., the degree of resolutiondesired); the age, weight, sex, and health and physical condition of thesubject to which the formulation is being administered; the route ofadministration; and the nature of the disease, disorder, condition orsymptom thereof. The dosing regimen may also take into consideration theexistence, nature, and extent of any adverse effects associated with theagent(s) being administered. Effective dosage amounts and dosageregimens can readily be determined from, for example, safety anddose-escalation trials, in vivo studies (e.g., animal models), and othermethods known to the skilled artisan. In general, dosing parametersdictate that the dosage amount be less than an amount that could beirreversibly toxic to the subject (the maximum tolerated dose (MID)) andnot less than an amount required to produce a measurable effect on thesubject. Such amounts are determined by, for example, thepharmacokinetic and pharmacodynamic parameters associated with ADME,taking into consideration the route of administration and other factors.

In some embodiments, an CD73 inhibitor may be administered (e.g.,orally) at dosage levels of about 0.01 mg/kg to about 50 mg/kg, or about1 mg/kg to about 25 mg/kg, of subject body weight per day, one or moretimes a day, to obtain the desired therapeutic effect. Foradministration of an oral agent, the compositions can be provided in theform of tablets, capsules and the like containing from 1.0 to 1000milligrams of the active ingredient, particularly 1, 3, 5, 10, 15, 20,25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, or1000 milligrams of the active ingredient.

In some embodiments, the dosage of the desired CD73 inhibitor iscontained in a “unit dosage form”. The phrase “unit dosage form” refersto physically discrete units, each unit containing a predeterminedamount of the CD73 inhibitor, either alone or in combination with one ormore additional agents, sufficient to produce the desired effect. Itwill be appreciated that the parameters of a unit dosage form willdepend on the particular agent(s) and the effect to be achieved.

There are also provided herein kits comprising a CD73 inhibitor compoundor composition. Kits are generally in the form of a physical structurehousing various components and may be used, for example, in practicingthe methods provided herein. For example, a kit may include one or moreCD73 inhibitor disclosed herein (provided in, e.g., a sterilecontainer), which may be in the form of a pharmaceutical compositionsuitable for administration to a subject. The CD73 inhibitor can beprovided in a form that is ready for use (e.g., a tablet or capsule) orin a form requiring, for example, reconstitution or dilution (e.g., apowder) prior to administration. When the CD73 inhibitors are in a formthat needs to be reconstituted or diluted by a user, the kit may alsoinclude diluents (e.g., sterile water), buffers, pharmaceuticallyacceptable excipients, and the like, packaged with or separately fromthe CD73 inhibitors. When combination therapy is contemplated, the kitmay contain several therapeutic agents separately or they may already becombined in the kit. Each component of the kit may be enclosed within anindividual container, and all of the various containers may be within asingle package. A kit of the present invention may be designed forconditions necessary to properly maintain the components housed therein(e.g., refrigeration or freezing).

A kit may also contain a label or packaging insert including identifyinginformation for the components therein and instructions for their use(e.g., dosing parameters, clinical pharmacology of the activeingredient(s), including mechanism of action, pharmacokinetics andpharmacodynamics, adverse effects, contraindications, etc.). Labels orinserts can include manufacturer information such as lot numbers andexpiration dates. The label or packaging insert may be, e.g., integratedinto the physical structure housing the components, contained separatelywithin the physical structure, or affixed to a component of the kit(e.g., an ampule, tube or vial).

Examples

The present invention will be more readily understood by referring tothe following examples, which are provided to illustrate the inventionand are not to be construed as limiting the scope thereof in any manner.

Unless defined otherwise or the context clearly dictates otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. It should be understood that any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention.

Compound Synthesis

Compounds provided herein can be prepared using conventional methods andas described in the Examples below.

The amine compound RNH₂ was obtained either from a commercial source, orprepared according to methods described in the literature.

Preparation of Triethylammonium Hydrogen Carbonate Buffer (TEAC).

A 1 M solution of TEAC was prepared by adding dry ice slowly to a 1 Mtriethylamine solution in water for several hours until the pH of thesolution reached approximately 7.4-7.6 (as measured using a pH meter).

The 2-chloropurine nucleoside derivative S-x (1 mmol, 1 eq.) wasdissolved in trimethyl phosphate (10 mL). The solution was cooled withan ice-bath. To the cold solution was added a solution ofbis(dichlorophosphoryl)methane (4.0 eq.) in trimethyl phosphate (5 mL).The resulting mixture was stirred at 0° C. for 2-4 h, and the reactionwas monitored by thin-layer chromatography (TLC). The reaction wasquenched by TEAC solution, and the pH of the reaction solution wasadjusted to 7-8. The mixture was extracted with dichloromethane (DCM)and the aqueous phase was isolated and concentrated. The residualmaterial was purified by reversed-phase chromatography with aC18-column, giving the product as colorless solid.

Example 1. Synthesis of Compound 1

DIEA (diisopropylethylamine; 7.5 mmol, 969 mg, 1.5 eq.) was addeddropwise to a solution of2,6-dichloro-9-(2′,3′,5′-tri-O-acetyl-β-D-ribofuranosyl)purine (5.0mmol, 2236 mg, 1.0 eq.) and benzylamine (5.0 mmol, 536 mg, 1.0 eq.) indioxane (25 mL). The reaction mixture was stirred at room temperatureovernight. The solvent was evaporated in vacuo and the residue wasdissolved in DCM (100 mL), washed with water (2×30 mL). The crudeproduct was purified by column chromatography. The intermediate wasdissolved in 50 mL NH₃/CH₃OH solution and stirred at 35° C. overnight.The solvent was evaporated in vacuo; and the residual material waspurified by column chromatography, giving 2-chloropurine nucleosidederivative S-1 (1818 mg).

S-1 (1.0 mmol, 392 mg, 1.0 eq.) was dissolved in trimethyl phosphate (10mL), cooled in an ice bath. To the cold solution was added a solution ofbis(dichlorophosphoryl)methane (4.0 eq.) in trimethyl phosphate (5 mL).The reaction mixture was stirred at 0° C. for 2-4 h; and the reactionwas monitored by TLC. The reaction was quenched by TEAC solution, andthe pH of the reaction solution was adjusted to 7-8. The mixture wasextracted with DCM and the aqueous phase was isolated, and concentrated.The residual material was purified by reversed-phase columnchromatography (C18-column), providing compound 1 as colorless solid(369 mg): ¹H NMR (500 MHz, CD₃OD-d₄) δ ppm 2.46 (t, 2H), 4.23-4.74 (m,7H), 6.01 (d, 1H), 7.19-7.38 (m, 5H), 8.59 (s, 1H); ¹³C NMR (125 MHz,CD₃Cl-d₃) δ ppm 40.12, 43.84, 63.92, 69.87, 74.64, 83.71, 88.50, 115.22,126.92, 127.47, 128.13, 149.27, 154.08, 154.96, 160.55; ³¹P NMR (200MHz, CD₃Cl-d₃) δ ppm 12.94, 18.11; m/z (ESI⁺) 550.1.

Example 2. Synthesis of Compound 6

DIEA (7.5 mmol, 969 mg, 1.5 eq.) was added dropwise to a solution of2,6-dichloro-9-(2′,3′,5′-tri-O-acetyl-β-D-ribofuranosyl)purine (5.0mmol, 2.2 g, 1.0 eq.) and 1-naphthalenemethanamine (5.0 mmol, 786 mg,1.0 eq.) in dioxane (25 mL). The reaction mixture was stirred at roomtemperature overnight. The solvent was evaporated in vacuo and theresidue was dissolved in DCM (100 mL), washed with water (2×30 mL).Solvent was removed (using a rotary evaporator), and the residualmaterial was purified by column chromatography. The intermediate wasdissolved in 50-mL NH₃/CH₃OH solution and stirred at 35° C. overnight.Solvent was evaporated in vacuo and the residual material was purifiedby column chromatography, giving S-6 (1.3 g).

S-6 (1.0 mmol, 442 mg, 1.0 eq.) was dissolved in trimethyl phosphate (10mL), cooled in an ice bath. To the cold solution was added a solution ofbis(dichlorophosphoryl)methane (4.0 eq.) in trimethyl phosphate (5 mL).The resulting mixture was stirred at 0° C. for 2-4 h; and the reactionwas monitored by TLC. The reaction was quenched by TEAC solution, andthe pH of the reaction mixture was adjusted to 7-8. The mixture wasextracted with DCM. The aqueous phase was isolated, concentrated; andthe residual material was purified by reversed-phase chromatography(C18-column), giving compound 6 as a colorless solid (110 mg): ¹H NMR(500 MHz, D₂O) δ ppm 0.88-0.91 (m, 3H), 1.34-1.43 (m, 4H), 1.67-1.68 (m,2H), 2.15-2.26 (m, 2H), 4.16-4.22 (m, 2H), 4.25-4.31 (m, 2H), 4.38-4.41(m, 1H), 4.53-4.57 (m, 1H), 4.74-4.76 (m, H), 6.13-1.15 (m, 1H),8.70-8.75 (m, 1H); ¹³C NMR (125 MHz, D₂O) δ ppm 13.20, 21.60, 27.24,27.55, 63.46, 67.12, 70.15, 74.34, 84.08, 84.14, 87.38, 120.98, 142.83,150.24, 152.49, 153.22; ³¹P NMR (200 MHz, D₂O) δ ppm 16.15, 18.97; m/z(ES⁻) 571.8.

Example 3. Synthesis of Compound 7

DIEA (7.5 mmol, 969 mg, 1.5 eq.) was added dropwise to a solution of2,6-dichloro-9-(2′,3′,5′-tri-O-acetyl-β-D-ribofuranosyl)purine (5.0mmol, 2.2 g, 1.0 eq.) and 2-naphthalenemethanamine (5.0 mmol, 786 mg,1.0 eq.) in dioxane (25 mL). The reaction was stirred at roomtemperature overnight. The solvent was evaporated in vacuo and theresidue was dissolved in DCM (100 mL), washed with water (2×30 mL). Thecrude product was purified by column chromatography. The intermediatewas dissolved in 50 mL NH₃/CH₃OH solution and stirred at 35° C.overnight. The solvent was evaporated in vacuo and the residual materialwas purified by column chromatography, giving 2-chloropurine nucleosidederivative S-7 (1.15 g).

S-7 (1.0 mmol, 442 mg, 1.0 eq.) was dissolved in trimethyl phosphate (10mL), and the solution was cooled with an ice-bath. To the cold solutionwas added a solution of bis(dichlorophosphoryl)methane (4.0 eq.) intrimethyl phosphate (5 mL). The reaction mixture was stirred at 0° C.for 2-4 h; and the reaction was monitored by TLC. The reaction wasquenched by TEAC solution, and the pH of the reaction mixture wasadjusted to 7-8. The mixture was extracted with DCM; and the aqueousphase was isolated and concentrated. The residual material was purifiedby reversed-phase chromatography (C18-column), providing compound 7 as acolorless solid (105 mg): ¹H NMR (500 MHz, D₂O) δ ppm 2.11 (t, J=19.7Hz, 2H), 4.10 (s, 2H), 4.29 (s, 1H), 4.42 (s, 1H), 4.55 (s, 1H), 4.88(s, 2H), 5.79 (s, 1H), 7.36-7.44 (m, 4H), 7.71 (d, J=7.9 Hz, 1H), 7.76(d, J=7.2 Hz, 1H), 7.87 (d, J=7.8 Hz, 1H), 8.13 (s, 1H); ¹³C NMR (125MHz, D₂O) δ ppm 27.57, 42.23, 63.39, 70.05, 74.16, 83.59, 86.92, 117.78,122.74, 125.48, 125.88, 126.05, 126.26, 128.24, 128.43, 130.48, 132.19,133.08, 139.02, 148.62, 153.86, 154.44. ³¹P NMR (202 MHz, D₂O) δ ppm15.17, 19.58; m/z (ES⁻) 598.2.

Example 4. Synthesis of Compound 8

The 2-chloropurine nucleoside derivative S-8 (1.0 mmol, 415 mg, 1.0 eq.)was dissolved in trimethyl phosphate (10 mL); and the solution wascooled with an ice-bath. To the cold solution was added a solution ofbis(dichlorophosphoryl)methane (4.0 eq.) in trimethyl phosphate (5 mL),while the ice bath was applied. The resulting mixture was stirred at 0°C. for 2-4 h; and the reaction was monitored by TCL. The reaction wasquenched by TEAC solution; and the pH of the reaction mixture wasadjusted to 7-8. The mixture was extracted with DCM; and the aqueousphase was isolated and concentrated. The residual material was purified(reversed-phase column chromatography, C18-column), to obtain theproduct as colorless solid (58 mg). ¹H NMR (500 MHz, D₂O) δ ppm 2.12 (t,J=19.7 Hz, 2H), 4.08 (s, 2H), 4.27 (s, 1H), 4.40 (s, 1H), 4.53 (s, 1H),4.65 (s, 2H), 5.75 (s, 1H), 7.36 (s, 3H), 7.65 (s, 2H), 7.69 (d, J=8.0Hz, 2H), 8.28 (s, 1H); ¹³C NMR (125 MHz, D₂O) δ ppm 44.04, 63.41, 70.06,74.19, 83.73, 86.90, 125.50, 125.60, 126.00, 126.34, 127.38, 128.15,132.13, 132.71, 135.18, 139.21, 153.96, 154.73; ³¹P NMR (202 MHz, D₂O) δppm 15.86, 19.01; m/z (ES⁻) 598.4.

Example 5. Synthesis of Compound 9

DIEA (12.5 mmol, 1.6 g, 2.5 eq.) was added dropwise to a solution of2,6-dichloro-9-(2′,3′,5′-tri-O-acetyl-β-D-ribofuranosyl)purine (5.0mmol, 2.2 g, 1.0 eq.) and memantine hydrochloride (5.0 mmol, 1.0 g, 1.0eq.) in 25-mL dioxane. The reaction mixture was stirred at roomtemperature overnight. The solvent was evaporated in vacuo and theresidue was dissolved in DCM (100 mL), washed with water (2×30 mL), andconcentrated. The residual material was purified by columnchromatography, to give the intermediate. The intermediate was dissolvedin 50 mL NH₃/CH₃OH solution and the mixture was stirred at 35° C.overnight. The solvent was evaporated in vacuo and the residual materialwas purified by column chromatography, giving 2-chloropurine nucleosidederivative S-9 (1.1 g).

S-9 (1.0 mmol, 463 mg, 1.0 eq.) was dissolved in trimethyl phosphate (10mL); and the mixture was cooled with an ice-bath. To the cold mixturewas added a solution of bis(dichlorophosphoryl)methane (4 eq.) intrimethyl phosphate (5 mL). The resulting mixture was stirred at 0° C.for 2-4 h; and the reaction was monitored by TLC. After the reactionfinished, the reaction was quenched by TEAC solution. The pH of themixture was adjusted to 7-8. The mixture was extracted with DCM and theaqueous phase was isolated. The aqueous solution was concentrated; andthe residue was purified by reversed-phase column chromatography (C18column), providing compound 9 as a colorless solid (200 mg): ¹H NMR (500MHz, D₂O) δ ppm 0.76 (s, 6H), 0.98-1.12 (m, 2H), 1.22 (s, 2H), 1.31 (d,J=11.3 Hz, 2H), 1.70 (dd, J=29.4, 11.9 Hz, 4H), 1.90 (s, 2H), 2.11 (t,J=19.9 Hz, 3H), 4.07 (s, 2H), 4.28 (s, 1H), 4.44 (dd, J=6.5, 2.4 Hz,1H), 4.67-4.63 (m, 1H), 5.91 (d, J=5.7 Hz, 1H), 8.33 (s, 1H); ¹³C NMR(125 MHz, D₂O) δ ppm 25.96, 26.95, 29.97, 32.08, 39.23, 42.32, 50.32,55.08, 63.73, 70.04, 74.33, 83.78, 87.45, 116.10, 138.34, 148.62,153.57, 154.16; ³¹P NMR (200 MHz, D₂O) δ ppm 18.12; m/z (ES⁻) 620.2.

Example 6. Synthesis of Compound 10

The 2-chloropurine nucleoside derivative S-10 (1.0 mmol, 451 mg, 1.0eq.) was dissolved in trimethyl phosphate (10 mL), and the solution wascooled in an ice-bath. To the cold solution was added a solution ofbis(dichlorophosphoryl)methane (4 eq.) in trimethyl phosphate (5 mL).The resulting mixture was stirred at 0° C. for 2-4 h, and the reactionwas monitored by TLC. The reaction was quenched by TEAC solution; andthe pH of the reaction solution was adjusted to 7-8. The mixture wasextracted with DCM and the aqueous phase was isolated and concentrated.The residual material was purified by reversed-phase columnchromatography (C18-column), giving compound 10 as a colorless solid (60mg): ¹H NMR (500 MHz, D₂O) δ ppm 2.27 (t, J=19.4 Hz, 2H), 4.17 (s, 2H),4.35 (s, 1H), 4.46 (s, 1H), 6.02 (s, 1H), 7.27 (s, 4H), 7.54 (s, 2H),7.97 (s, 2H), 8.33 (s, 1H); ¹³C NMR (125 MHz, D₂O) δ ppm 16.73, 25.47,26.48, 27.50, 57.39, 63.69, 70.09, 74.31, 83.84, 87.70, 113.16, 120.00,122.84, 124.66, 126.42, 138.03, 143.08, 148.86, 152.70, 154.12; ³¹P NMR(200 MHz, D₂O) δ ppm 17.43, 19.35-19.97; m/z (ES⁻) 608.0.

Example 7. Synthesis of Compound 11

The 2-Chloropurine nucleoside derivative S-11 (1.0 mmol, 564 mg, 1.0eq.) was dissolved in trimethyl phosphate (10 mL). The solution wascooled in an ice-bath; and to the cold solution was added a solution ofbis(dichlorophosphoryl)methane (4 eq.) in trimethyl phosphate (5 mL).The resulting mixture was stirred at 0° C. for 2-4 h; and the reactionwas monitored by TLC. After being quenched by TEAC solution, the pH ofthe reaction mixture was adjusted to 7-8. This mixture was extractedwith DCM and the aqueous phase was isolated and concentrated. Theresidual material was purified by reversed-phase column chromatography(C18-column), providing compound 11 as a colorless solid (100 mg): ¹HNMR (500 MHz, D₂O) δ ppm 0.77 (d, J=6.5 Hz, 3H), 1.22 (s, 14H), 1.55 (s,2H), 1.92 (s, 4H), 2.12 (t, J=19.8 Hz, 2H), 2.67 (d, J=38.2 Hz, 4H),4.03 (s, 2H), 4.19 (s, 1H), 4.40 (s, 1H), 4.60 (s, 1H), 5.20 (d, J=5.6Hz, 4H), 5.88 (d, J=4.4 Hz, 1H), 8.51 (s, 1H); ¹³C NMR (125 MHz, D₂O) δppm 13.90, 22.48, 24.71, 25.52, 27.10, 29.26, 29.64, 31.41, 36.86,37.49, 63.89, 70.44, 74.37, 84.15, 86.83, 119.95, 127.82, 129.70,149.34, 152.45, 152.86, 164.88, 174.66; ³¹P NMR (200 MHz, D₂O) δ ppm16.04, 18.65; m/z (ES⁻) 720.4.

Example 8. Synthesis of Compound 12

DIEA (7.5 mmol, 969 mg, 1.5 eq.) was added dropwise to a solution of2,6-dichloro-9-(2′,3′,5′-tri-O-acetyl-β-D-ribofuranosyl)purine (5.0mmol, 2.23 g, 1.0 eq.) and 2-aminonaphthalene (5.0 mmol, 715 mg, 1.0eq.) in 25-mL dioxane. The reaction mixture was stirred at roomtemperature overnight. The solvent was evaporated in vacuo; and theresidue was dissolved in DCM (100 mL), washed with water (2×30 mL). TheDCM solution was concentrated to dryness; and the residual material waspurified by column chromatography, giving an intermediate. Theintermediate was dissolved in 50 mL NH₃/CH₃OH solution and the mixturewas stirred at 35° C. overnight. The solvent was evaporated in vacuo andthe residual material was purified by column chromatography, providingcompound S-12 (670 mg).

S-12 (1.0 mmol, 427 mg, 1.0 eq.) was dissolved in trimethyl phosphate(10 mL), and the solution was cooled in an ice-bath. To the coldsolution was added a solution of bis(dichlorophosphoryl)methane (4 eq.)in trimethyl phosphate (5 mL). The resulting mixture was stirred at 0°C. for 2-4 h; and the reaction was monitored by TLC. The reaction wasquenched by TEAC solution, and the pH of the quenched reaction mixturewas adjusted to 7-8. The mixture was extracted with DCM; and the aqueousphase was isolated and concentrated. The residual material was purifiedby reversed-phase column chromatography (C18-column), giving compound 12as a colorless solid (100 mg): ¹H NMR (D₂O, 500 MHz) δ ppm 2.21-2.29 (m,2H), 4.20-4.22 (m, 2H), 4.40-4.44 (m, 1H), 4.56-4.60 (m, 1H), 4.78-4.80(m, 1H), 6.09-6.10 (m, 1H), 7.58-7.64 (m, 3H), 7.71-7.73 (m, 1H),7.97-7.99 (m, 1H) 8.02-8.04 (m, 2H), 8.55 (s, 1H); ¹³C NMR (D₂O, 125MHz) δ ppm 26.38, 27.37, 28.36, 63.61, 70.33, 84.01, 86.76, 118.06,121.57, 122.25, 125.52, 126.23, 126.41, 139.82, 149.74, 153.39, 153.47;³¹P NMR (D₂O, 200 MHz) δ ppm 16.14, 18.96; m/z (ES⁻) 583.9.

Example 9. Synthesis of Compound 15

The 2-Chloropurine nucleoside derivative S-15 (1.0 mmol, 513 mg, 1.0eq.) was dissolved in trimethyl phosphate (10 mL). The mixture wascooled in an ice-bath, followed by addition of a solution ofbis(dichlorophosphoryl)methane (4 eq.) in trimethyl phosphate (5 mL).The resulting mixture was stirred at 0° C. for 2-4 h, and the reactionwas monitored by TLC. The reaction was quenched by TEAC solution; andthe pH of the reaction mixture was adjusted to 7-8. The mixture wasextracted with DCM; and the aqueous phase was isolated and concentrated.The residual material was purified by reversed-phase columnchromatography (C18-column), giving compound 15 as a colorless solid (30mg): ¹H NMR (500 MHz, D₂O) δ ppm 0.70 (t, J=6.3 Hz, 3H), 1.06 (s, 16H),1.27 (s, 2H), 1.58 (s, 2H), 2.10 (t, J=19.7 Hz, 2H), 4.12 (d, J=32.1 Hz,4H), 4.27 (s, 1H), 4.46 (s, 1H), 4.66 (s, 1H), 6.00 (d, J=4.7 Hz, 1H),8.60 (s, 1H); ¹³C NMR (125 MHz, D₂O) δ ppm 13.73, 22.40, 25.51, 27.55,28.40, 29.07, 29.36, 31.66, 63.64, 66.70, 70.27, 74.36, 83.95, 87.18,120.57, 142.65, 150.05, 152.38, 153.21; ³¹P NMR (200 MHz, D₂O) δ ppm15.64, 18.89; m/z (ES⁻) 670.1.

Example 10. Synthesis of Compound 16

The 2-Chloropurine nucleoside derivative S-16 (1.0 mmol, 478 mg, 1.0eq.) was dissolved in trimethyl phosphate (10 mL); and the mixture wascooled in an ice-bath, followed by addition of a solution ofbis(dichlorophosphoryl)methane (4 eq.) in trimethyl phosphate (5 mL).The resulting mixture was stirred at 0° C. for 2-4 h; and the reactionwas monitored by TLC. The reaction was quenched by TEAC solution; andthe pH of the mixture was adjusted to 7-8. The mixture was extractedwith DCM; and the aqueous phase was isolated and concentrated. Theresidual material was purified by reversed-phase column chromatography(C18-column), providing compound 16 as a colorless solid (250 mg): ¹HNMR (500 MHz, D₂O) δ ppm 1.64 (s, 11H), 1.91 (s, 4H), 2.16 (t, J=19.8Hz, 2H), 2.29 (s, 2H), 4.14 (dddd, J=6.8, 5.9, 4.1, 1.6 Hz, 3H),4.30-4.37 (m, 1H), 4.56-4.46 (m, 1H), 6.11 (d, J=5.1 Hz, 1H), 8.72 (s,1H); ¹³C NMR (125 MHz, D₂O) δ ppm 16.75, 27.23, 28.38, 33.41, 36.07,42.03, 51.44, 63.47, 70.17, 74.37, 84.19, 87.49, 115.91, 121.90, 143.36,149.28, 153.15, 173.66; ³¹P NMR (200 MHz, D₂O) δ ppm 16.37, 18.85; m/z(ES⁻) 634.1.

Example 11. Synthesis of Compound 17

DIEA (12.5 mmol, 1.6 g, 2.5 eq.) was added dropwise to a solution of2,6-dichloro-9-(2′,3′,5′-tri-O-acetyl-β-D-ribofuranosyl)purine (5.0mmol, 2.23 g, 1.0 eq.) and 2-adamantanamine hydrochloride (5.0 mmol,0.94 g, 1.0 eq.) in 25-mL dioxane. The mixture was stirred at roomtemperature overnight. The solvent was evaporated in vacuo and theresidue was dissolved in DCM (100 mL), washed with water (2×30 mL). Theorganic layer was concentrated; and the residual material was purifiedby column chromatography, giving an intermediate. This intermediate wasdissolved in 50 mL NH₃/CH₃OH solution and stirred at 35° C. overnight.After evaporation of the solvent, the residual material was purified bycolumn chromatography, providing 2-chloropurine nucleoside derivativeS-17 (880 mg). Compound S-17 (1.0 mmol, 435 mg, 1.0 eq.) was dissolvedin trimethyl phosphate (10 mL); and the mixture was cooled in anice-bath. To the cold mixture was added a solution ofbis(dichlorophosphoryl)methane (4 eq.) in trimethyl phosphate (5 mL).The resulting mixture was stirred at 0° C. for 2-4 h; and the reactionwas monitored by TLC. The reaction was quenched by TEAC solution; andthe pH of the mixture was adjusted to 7-8. The mixture was extractedwith DCM; and the aqueous phase was isolated and concentrated. Theresidual material was purified by reversed-phase column chromatography(C18-column), giving compound 17 as a colorless solid (30 mg): ¹H NMR(500 MHz, D₂O) δ ppm 1.60 (d, J=12.7 Hz, 2H), 1.71 (s, 2H), 1.82 (d,J=20.8 Hz, 7H), 1.96 (d, J=19.6 Hz, 4H), 2.14 (t, J=19.8 Hz, 2H), 4.10(s, 2H), 4.20 (s, 1H), 4.31 (s, 1H), 4.47 (t, J=4.2 Hz, 1H), 5.96 (d,J=5.6 Hz, 1H), 8.41 (s, 1H); ¹³C NMR (125 MHz, D₂O) δ ppm 16.70, 26.77,30.81, 31.44, 36.48, 36.86, 57.36, 63.51, 70.23, 74.20, 83.97, 86.73,139.05, 154.39; ³¹P NMR (200 MHz, D₂O) δ ppm 15.85, 19.04; m/z (ES⁻)592.0.

Example 12. Synthesis of Compound 18

The 2-Chloropurine nucleoside derivative S-18 (1.0 mmol, 512 mg, 1.0eq.) was dissolved in trimethyl phosphate (10 mL). The mixture wascooled in an ice-bath, followed by addition of a solution ofbis(dichlorophosphoryl)methane (4 eq.) in trimethyl phosphate (5 mL).The resulting mixture was stirred at 0° C. for 2-4 h; and the reactionwas monitored by thin layer chromatography. The reaction was quenched byTEAC solution; and the pH of the mixture was adjusted to 7-8. Themixture was extracted with DCM; and the aqueous phase was isolated andconcentrated. The residual material was purified by reversed-phasecolumn chromatography (C18-column), giving compound 18 as a colorlesssolid (70 mg): ¹H NMR (500 MHz, CD₃OD-d₄) δ ppm 0.93 (t, J=6.8 Hz, 3H),1.52 (s, 18H), 1.60-1.74 (m, 2H), 2.33 (t, J=19.8 Hz, 2H), 3.43 (t,J=6.6 Hz, 2H), 4.27 (d, J=21.4 Hz, 3H), 4.47-4.61 (m, 1H), 4.71 (t,J=5.3 Hz, 1H), 6.13 (d, J=5.4 Hz, 1H), 8.76 (s, 1H); ¹³C NMR (125 MHz,CD₃OD-d₄) δ ppm 13.01, 22.30, 26.62, 28.98, 29.28, 31.63, 39.56, 63.95,70.57, 74.91, 84.44, 87.86, 118.78, 142.32, 150.79, 151.78, 151.97,154.17; ³¹P NMR (200 MHz, CD₃OD-d₄) δ ppm 16.03, 20.25; m/z (ES⁻),669.2.

Example 13. Synthesis of Compound 19

DIEA (7.5 mmol, 969 mg, 1.5 eq.) was added dropwise to a solution of2,6-dichloro-9-(2′,3′,5′-tri-O-acetyl-β-D-ribofuranosyl)purine (5.0mmol, 2.23 g, 1.0 eq.) and di-N-dodecylamine (5.0 mmol, 1.8 g, 1.0 eq.)in 25 mL dioxane. The reaction mixture was stirred at room temperatureovernight. The solvent was evaporated in vacuo; and the residue wasdissolved in DCM (100 mL), washed with water (2×30 mL). The organiclayer was concentrated to dryness; and the residual material waspurified by column chromatography, giving an intermediate compound. Thisintermediate was dissolved in 50 mL NH₃/CH₃OH solution and the mixturewas stirred at 35° C. overnight. After removal of solvent in vacuo, theresidual material product was purified by column chromatography, giving2-chloropurine nucleoside derivative S-19 (1.3 g).

S-19 (1.0 mmol, 637 mg, 1.0 eq.) was dissolved in trimethyl phosphate(10 mL). The mixture was cooled in an ice-bath, followed by addition ofa solution of bis(dichlorophosphoryl)methane (4 eq.) in trimethylphosphate (5 mL). The resulting mixture was stirred at 0° C. for 2-4 h;and the reaction was monitored by TLC. The reaction was quenched by TEACsolution; and the pH of the reaction solution was adjusted to 7-8. Themixture was extracted with DCM; and the aqueous phase was isolated andconcentrated. The residual material was purified by reversed-phasecolumn chromatography (C18-column), providing compound 19 as a colorlesssolid (150 mg): ¹H NMR (500 MHz, CD₃OD-d₄) δ ppm 0.93 (t, J=6.6 Hz, 6H),1.32-1.46 (m, 36H), 1.72 (s, 4H), 2.36 (t, J=20.0 Hz, 2H), 3.70 (s, 2H),4.11-4.32 (m, 5H), 4.47 (s, 1H), 4.66 (t, J=5.2 Hz, 1H), 6.04 (d, J=5.4Hz, 1H), 8.37 (s, 1H); ¹³C NMR (125 MHz, CD₃OD-d₄) δ ppm 13.08, 22.34,26.44, 29.08, 29.34, 31.68, 64.24, 70.51, 74.51, 83.84, 87.46, 118.27,137.84, 151.64, 153.46, 154.25; ³¹P NMR (200 MHz, CD₃OD-d₄) δ ppm 16.20,19.99; m/z (ES⁻) 794.6.

Example 14. Synthesis of Compound 20

DIEA (7.5 mmol, 969 mg, 1.5 eq.) was added dropwise to a solution of2,6-dichloro-9-(2′,3′,5′-tri-O-acetyl-β-D-ribofuranosyl)purine (5.0mmol, 2.23 g, 1.0 eq.) and 2-anthracenamine (5.0 mmol, 1.0 g, 1.0 eq.)in 25 mL dioxane. The reaction mixture was stirred at room temperatureovernight. The solvent was evaporated in vacuo and the residue wasdissolved in DCM (100 mL), washed with water (2×30 mL). The organiclayer was concentrated to dryness; and the residual material waspurified by column chromatography, giving an intermediate compound. Thisintermediate was dissolved in 50 mL NH₃/CH₃OH solution and stirred at35° C. overnight. The solvent was evaporated in vacuo and the residualmaterial was purified by column chromatography, giving 2-chloropurinenucleoside derivative S-20 (770 mg).

S-20 (1.0 mmol, 477 mg, 1.0 eq.) was dissolved in trimethyl phosphate(10 mL). The mixture was cooled in an ice-bath, followed by addition ofa solution of bis(dichlorophosphoryl)methane (4 eq.) in trimethylphosphate (5 mL). The resulting mixture was stirred at 0° C. for 2-4 h;and the reaction was monitored by TLC. The reaction was quenched by TEACsolution; and the pH of the reaction mixture was adjusted to 7-8. Themixture was extracted with DCM; and the aqueous phase was isolated andconcentrated. The residual material was purified by reversed-phasecolumn chromatography (C18-column), providing compound 20 as a colorlesssolid (80 mg): ¹H NMR (500 MHz, D₂O) δ ppm 2.25 (t, J=19.4 Hz, 2H), 4.28(d, J=58.1 Hz, 3H), 4.46 (s, 2H), 5.56 (s, 1H), 7.19 (s, 3H), 7.57 (d,J=73.7 Hz, 4H), 7.85 (s, 2H), 8.21 (s, 1H); ³¹P NMR (200 MHz, D₂O) δ ppm18.42, 19.15; m/z (ES⁻) 633.9.

Example 15. Synthesis of Compound 22

DIEA (7.5 mmol, 969 mg, 1.5 eq.) was added dropwise to a solution of2,6-dichloro-9-(2′,3′,5′-tri-O-acetyl-β-D-ribofuranosyl)purine (5.0mmol, 2.23 g, 1.0 eq.) and 1-adamantanamin (5.0 mmol, 756 mg, 1.0 eq.)in 25 mL dioxane. The reaction mixture was stirred at room temperatureovernight. The solvent was evaporated in vacuo and the residue wasdissolved in DCM (100 mL), washed with water (2×30 mL). After removal ofsolvent, the residual material was purified by column chromatography,providing an intermediate compound. This intermediate was dissolved in50 mL NH₃/CH₃OH solution and the mixture was stirred at 35° C.overnight. The solvent was evaporated in vacuo and the residual materialwas purified by column chromatography, giving 2-chloropurine nucleosidederivative S-22 (770 mg).

S-22 (1.0 mmol, 435 mg, 1.0 eq.) was dissolved in trimethyl phosphate(10 mL). The mixture was cooled in an ice-bath, followed by addition ofa solution of bis(dichlorophosphoryl)methane (4 eq.) in trimethylphosphate (5 mL). The resulting mixture was stirred at 0° C. for 2-4 h;and the reaction was monitored by TLC. The reaction was quenched by TEACsolution; and the pH of the reaction solution was adjusted to 7-8. Themixture was extracted with DCM; and the aqueous phase was isolated andconcentrated. The residual material was purified by reversed phasecolumn chromatography (C18-column), giving compound 22 as a colorlesssolid (100 mg): ¹H NMR (500 MHz, D₂O) δ ppm 1.64 (s, 6H), 2.06 (d,J=33.5 Hz, 9H), 2.17 (d, J=19.9 Hz, 2H), 4.09 (s, 2H), 4.30 (d, J=0.9Hz, 1H), 4.45 (ddd, J=5.9, 3.0, 2.0 Hz, 1H), 4.68-4.65 (m, 1H), 5.94 (d,J=5.2 Hz, 1H), 8.38 (s, 1H); ¹³C NMR (125 MHz, D₂O) δ ppm 26.22, 27.22,29.28, 35.72, 40.80, 53.50, 63.57, 70.19, 74.20, 83.95, 86.81, 138.60,148.74, 153.76, 154.43; ³¹P NMR (200 MHz, D₂O) δ ppm 16.38, 18.81; m/z(ES⁻) 592.2.

Example 16 Synthesis of Compound 23

The 2-Chloropurine nucleoside derivative S-23 (1.0 mmol, 481 mg, 1.0eq.) was dissolved in trimethyl phosphate (10 mL), and the mixture wascooled in an ice-bath. To the cold mixture was added a solution ofbis(dichlorophosphoryl)methane (4 eq.) in trimethyl phosphate (5 mL).The resulting mixture was stirred at 0° C. for 2-4 h; and the reactionwas monitored by TLC. The reaction was quenched by TEAC solution; andthe pH of the reaction mixture was adjusted to 7-8. The mixture wasextracted with DCM; and the aqueous phase was isolated and concentrated.The residual material was purified by reversed-phase columnchromatography (C18-column), giving compound 23 as colorless solid (141mg): ¹H NMR (D₂O, 500 MHz) δ ppm 0.90-0.97 (m, 9H), 1.18-1.20 (m, 1H),1.36-1.40 (m, 1H), 1.75-1.80 (m, 1H), 1.90-1.97 (m, 1H), 2.00-2.30 (m,2H), 2.40-2.45 (m, 1H), 4.20-4.25 (m, 2H), 4.40-4.44 (m, 1H), 4.44-4.57(m, 1H), 4.72-4.73 (m, 1H), 4.91-5.05 (m, 1H), 6.43-6.45 (m, 1H), 9.02(m, 1H); ¹³C NMR (D₂O, 125 MHz) δ 12.74, 18.03, 18.90, 35.91, 44.47,47.41, 48.45, 63.50, 70.16, 74.34, 83.22, 84.06, 84.13, 87.37, 120.84,142.72, 150.23, 152.41, 153.24, 153.45 ppm; ³¹P NMR (D₂O, 200 MHz) δ ppm16.49, 18.79; m/z (ES⁻) 638.0.

Example 17. Synthesis of Compound 31

DIEA (7.5 mmol, 969 mg, 1.5 eq.) was added dropwise to a solution of2,6-dichloro-9-(2′,3′,5′-tri-O-acetyl-β-D-ribofuranosyl)purine (5.0mmol, 2.23 g, 1.0 eq.) and 3-azaspiro[4.5]decane (5.0 mmol, 696 mg, 1.0eq.) in 25 mL dioxane. The reaction mixture was stirred at roomtemperature overnight. Solvent was evaporated in vacuo and the residuewas dissolved in DCM (100 mL), washed with water (2×30 mL). The organiclayer was evaporated to dryness, and the residual material was purifiedby column chromatography, providing an intermediate compound. Thisintermediate was dissolved in 50 mL NH₃/CH₃OH solution and stirred at35° C. overnight. Solvent was evaporated in vacuo; and the residualmaterial was purified by column chromatography, giving the corresponding2-chloropurine nucleoside derivative, S-31 (1.1 g).

S-31 (1.0 mmol, 423 mg, 1.0 eq.) was dissolved in trimethyl phosphate(10 mL); and the mixture was cooled in an ice-bath. To the cold mixturewas added a solution of bis(dichlorophosphoryl)methane (4 eq.) intrimethyl phosphate (5 mL). The resulting mixture was stirred at 0° C.for 2-4 h; and the reaction was monitored by TLC. The reaction wasquenched by TEAC solution; and the pH of the mixture was adjusted to7-8. The mixture was extracted with DCM; and the aqueous phase wascollected and concentrated. The residual material was purified byreversed-phase column chromatography (C18-column), giving compound 31 asa colorless solid (140 mg): ¹H NMR (500 MHz, D₂O) δ ppm 1.43 (t, J=22.8Hz, 10H), 1.83 (d, J=52.0 Hz, 2H), 2.15 (t, J=19.7 Hz, 2H), 3.38 (s,1H), 3.58 (s, 1H), 3.79 (s, 1H), 40.1 (s, 1H), 4.12 (s, 2H), 4.32 (s,1H), 4.49 (t, J=4.4 Hz, 1H), 4.67-4.70 (m, 1H), 5.97-5.98 (d, J=5.4 Hz,1H), 8.36-8.37 (d, J=6.8 Hz, 1H); ¹³C NMR (125 MHz, D₂O) δ ppm 7.67,8.70, 9.72, 22.92, 27.41, 34.49, 45.49, 47.78, 69.61, 73.58, 74.79,86.08, 87.43, 118.15, 139.22, 150.08, 153.06, 153.84; ³¹P NMR (200 MHz,D₂O) δ ppm 15.71, 19.11; m/z (ES⁻) 580.0.

Example 18. Synthesis of Compound 51

DIEA (7.5 mmol, 969 mg, 1.5 eq.) was added dropwise to a solution of2,6-dichloro-9-(2′,3′,5′-tri-O-acetyl-β-D-ribofuranosyl)purine (5.0mmol, 2.23 g, 1.0 eq.) and nortropine (5.0 mmol, 636 mg, 1.0 eq.) in 25mL dioxane. The reaction mixture was stirred at room temperatureovernight. The solvent was evaporated in vacuo and the residue wasdissolved in DCM (100 mL), washed with water (2×30 mL). The residualmaterial was purified by column chromatography, giving an intermediatecompound. This intermediate was dissolved in 50 mL NH₃/CH₃OH solution,and the mixture was stirred at 35° C. overnight. After removal ofsolvent (in vacuo), the residual material was purified by columnchromatography, giving a derivative of 2-chloropurine nucleoside S-51(1.2 g).

Compound S-51 (1.0 mmol, 411 mg, 1.0 eq.) was dissolved in trimethylphosphate (10 mL). The mixture was cooled in an ice-bath, followed byaddition of bis(dichlorophosphoryl)methane (4 eq.) in trimethylphosphate (5 mL). The resulting mixture was stirred at 0° C. for 2-4 h;and the reaction was monitored by TLC. The reaction was quenched by TEACsolution; and the pH of the reaction solution was adjusted to 7-8. Themixture was extracted then with DCM; and the aqueous phase was isolatedand concentrated. The residual material was purified by reversed-phasecolumn chromatography (C18-column), giving compound 51 as a colorlesssolid (160 mg): ¹H NMR (500 MHz, D₂O) δ ppm 1.78-2.42 (m, 11H), 4.06 (s,1H), 4.12 (s, 2H), 4.28-4.37 (m, 1H), 4.45-4.55 (m, 1H), 4.82-4.88 (m,1H), 5.38 (dd, J=3.4, 2.4 Hz, 1H), 5.99 (d, J=5.6 Hz, 1H), 8.40 (s, 1H);¹³C NMR (125 MHz, D₂O) δ ppm 26.05, 27.10, 27.68, 37.08, 38.20, 53.79,54.40, 64.38, 70.20, 74.09, 83.99, 86.71, 117.78, 138.54, 151.12,154.22; ³¹P NMR (200 MHz, D₂O) δ ppm 16.90, 18.63; m/z (ES⁻) 568.0.

Example 19. Synthesis of Compound a-1

In a 1000 ml round-bottomed flask was added D-Ribose (50 g, 333.05 mmol,1 eq.), Acetone (400 mL), 2,2-dimethoxypropane (100 mL) and HClO₄ (25 g,25.00 mL, 70% purity). The mixture was stirred at room temperature for2.5 h, followed by addition of a solution of MeOH (30 mL) and themixture was stirred overnight. After the completion of the reaction, itwas cooled to −30° C., then a solution of 30% Na₂CO₃ (75 mL) was slowlyadded so that the temperature did not exceed 10° C. The Acetone (400 mL)was removed to afford the crude product. The precipitate was filteredand washed with ethyl acetate (50 mL). The filtrate was concentrated.The resulting liquid was diluted with ethyl acetate (300 mL). Theorganic layer was washed with brine (300 mL), dried (Na₂SO₄), filteredand evaporated to dryness. The residue was purified by columnchromatography on silica gel eluted with (PE/EA from 100:0 to 70:30) togive[(3aR,4R,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]methanol(43 g, 63.22%). ¹H NMR (500 MHz, CDCl₃) δ ppm 1.29 (s, 3H), 1.44 (d,J=8.3 Hz, 3H), 3.36-3.45 (m, 3H), 3.52-3.72 (m, 2H), 4.39 (t, J=2.8 Hz,1H), 4.56 (d, J=5.9 Hz, 1H), 4.80 (d, J=5.9 Hz, 1H), 4.94 (s, 1H).

To a solution of[(3aR,4R,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]methanol(20 g, 97.93 mmol, 1 eq.) in DCM (700 mL) was added pyridine (17.82 g,225.25 mmol, 18.13 mL, 2.3 eq.). The mixture was cooled to about −10° C.Then trifluoromethanesulfonic anhydride (58.03 g, 205.66 mmol, 34.60 mL,2.1 eq.) was added dropwise into the mixture at about −10° C. Themixture become red and a lot of solid was formed, and kept at −10° C.for about 4 h. The organic layer was washed with water (400 mL),followed by brine (300 mL), dried with Na₂SO₄, filtered, and evaporatedto dryness. The residue was purified by column chromatography on silicagel eluted with (PE/EA from 100:0 to 85:15) to give[(3aR,4R,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]methyl trifluoromethanesulfonate (21.6 g, 65.59%) as a brownoil.

To a solution of 1-[ethoxy(methyl)phosphoryl]oxyethane (9.77 g, 64.23mmol, 1.0 eq.) in THF (60 mL) was cooled to about −78° C. under N₂protection. Then n-BuLi (2.5 M, 28.26 mL, 1.1 eq.) was added dropwiseinto the mixture at about −78° C. The mixture was stirred at −78° C. for25 min. The solution of[(3aR,4R,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]methyltrifluoromethanesulfonate (21.6 g, 64.23 mmol, 1 eq.) in THF (20 mL) wasadded dropwise into the mixture at about −78° C. The mixture was stirredat −78° C. for 1 h and then quenched by aq. NH₄Cl (60 mL) at about −78°C. H₂O (60 mL) and EtOAc (60 mL) were added into the mixture. Theorganic layer was separated and the water layer was extracted by EtOAc(60 mL). Combined the organic layer and concentrated and the residue waspurified by column chromatography on silica gel eluted with (PE/EA from100:0 to 0:100) to afford(3aR,4R,6R,6aR)-6-(2-diethoxyphosphorylethyl)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxole(11 g, 50.62%) as a yellow oil. ¹H NMR (500 MHz, CDCl₃) δ ppm: 1.28-1.34(m, 9H). 1.47 (s, 3H), 1.72-2.00 (m, 4H), 3.35 (t, J=10.7 Hz, 3H),4.01-4.19 (m, 5H), 4.53 (d, J=5.9 Hz, 1H), 4.60 (d, J=5.3 Hz, 1H), 4.94(s, 1H).

A solution of 2-[isopropoxy(methyl)phosphoryl]oxypropane (1.60 g, 8.87mmol, 2 eq) in THF (6 mL) was cooled to about −78° C. under N₂protection. n-BuLi (2.5 M, 4.43 mL, 2.5 eq.) was added dropwise into themixture at about −78° C. The mixture was stirred at −78° C. for 1 h.(3aR,6R,6aR)-6-(2-diethoxyphosphorylethyl)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxole(1.5 g, 4.43 mmol, 1 eq) in THF (5 mL) was added into the mixture atabout −78° C. The mixture was stirred at −78° C. for 10 minutes and roomtemperature for 2 h. The mixture was quenched by aq.NH₄Cl at roomtemperature. The organic layer was separated and the water layer wasextracted by EtOAc (50 mL). Combined the organic layer and concentratedto dry, the residue was purified by column chromatography on silica geleluted with (EA/MeOH from 100:0 to 90:10) to afford(3aR,6R,6aR)-6-[2-[diisopropoxyphosphorylmethyl(ethoxy)phosphoryl]ethyl]-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxole (1 g, 47.74%). ¹H NMR (500 MHz, CDCl₃): δ ppm 1.25-1.40 (m,21H), 1.80-1.82 (m, 2H), 1.94-2.05 (m, 2H), 2.28-2.32 (m, 2H), 3.27-3.29(m, 3H), 4.05-4.09 (m, 3H), 4.51-4.54 (m, 2H), 4.68-4.70 (m, 2H),4.86-4.89 (m, 1H).

A solution of (2R,3S,4R,5R)-2-[2-[diisopropoxyphosphorylmethyl(ethoxy)phosphoryl]ethyl]-5-methoxy-tetrahydrofuran-3,4-diol (1.7 g, 3.93 mmol,1 eq.) in 1,4-dioxane (7.6 mL) and aq. sulfuric acid (1 M, 19 mL, 4.83eq.) was heated to reflux for 2.5 h. After the reaction was cooled toroom temperature, it was adjusted to pH 7 by saturated NaHCO₃ (38 mL)and concentrated to dryness. The residue was evaporated with dry THFtwice to afford the crude product, which was used in the next stepwithout purification.

To the above solution of(3R,4S,5R)-5-[2-[diisopropoxyphosphorylmethyl(ethoxy)phosphoryl]ethyl]tetrahydrofuran-2,3,4-triol in dry pyridine (25 mL) wasadded dropwise acetic anhydride (5.0 mL) at 0° C. After the reactionmixture was stirred at room temperature for 16 h, it was evaporated todryness. The residue was adjusted to pH 2 by aq. HCl (1 M, 15 mL). Itwas treated with DCM (50 mL) and the organic layer was separated, washedwith brine (10 mL), dried, and concentrated. The residue was purified bycolumn chromatography on silica gel eluted with (DCM/MeOH from 100:0 to90:10) to give [(2R,3R,4R)-4,5-diacetoxy-2-[2-[diisopropoxyphosphorylmethyl(ethoxy)phosphoryl]ethyl]tetrahydrofuran-3-yl] acetate (1.5 g,70.28%) as a yellow oil. ¹H NMR (500 MHz, MeOD) δ ppm 1.34 (dt, J=12.6,6.4, 15H), 1.93-2.17 (m, 13H), 2.51-2.83 (m, 2H), 4.13 (dd, J=16.8, 7.2,2H), 4.23 (d, J=15.6, 1H), 4.66-4.80 (m, 2H), 5.06-5.21 (m, 1H), 5.30(t, J=15.2, 1H), 5.95-6.45 (m, 1H).

94 To a solution of[(2R,3R,4R)-4,5-diacetoxy-2-[2-[diisopropoxyphosphorylmethyl(ethoxy)phosphoryl]ethyl]tetrahydrofuran-3-yl]acetate (2.5 g, 4.59 mmol, 1 eq.) and 2,6-dichloro-9H-purine (911.2 mg,4.64 mmol, 1.05 eq.) in ACN (25 mL) was added DBU (768.7 mg, 5.05 mmol,572.93 μL, 1.1 eq.) and TMSOTf (2.25 g, 10.10 mmol, 1.83 mL, 2.2 eq.) at−10° C. The mixture was stirred at 50° C. for 1 h. The reactioncompletion was detected by TLC (EA/MeOH=20:1). The mixture wasconcentrated and purified by column chromatography on silica gel elutedwith (EA/MeOH from 100:0 to 95:5) to give[(2R,3R,4R,5R)-4-acetoxy-5-(2,6-dichloropurin-9-yl)-2-[2-[diisopropoxyphosphorylmethyl(ethoxy)phosphoryl]ethyl]tetrahydrofuran-3-yl] acetate (1.5 g, 63.83%). ¹H NMR (500 MHz, CDCl₃):δ ppm 1.21-1.31 (m, 15H), 1.65-2.06 (m, 10H), 2.35-2.46 (m, 2H),4.07-4.14 (m, 2H), 4.72-5.16 (m, 3H), 5.28-5.77 (m, 1H), 6.08-6.11 (m,1H), 8.40-9.20 (m, 1H).

To a solution of[(2R,3R,4R,5R)-4-acetoxy-5-(2,6-dichloropurin-9-yl)-2-[2-[diisopropoxyphosphorylmethyl(ethoxy)phosphoryl]ethyl]tetrahydrofuran-3-yl] acetate(250 mg, 371.24 μmol, 1 eq.) and phenylmethanamine (47.74 mg, 445.49μmol, 1.2 eq.) in 1,4-dioxane (2.5 mL) was added DIPEA (143.94 mg, 1.11mmol, 193.99 μL, 3 eq.). The mixture was stirred at 120° C. for 16 h.The reaction was complete detected by TLC (EtOAc:MeOH=10:1). The mixturewas concentrated and purified by column chromatograph on silica geleluted with (EA/MeOH from 100:0 to 90:10) to give[(2R,3R,4R,5R)-4-acetoxy-5-[6-(benzylamino)-2-chloro-purin-9-yl]-2-[2-[diisopropoxyphosphorylmethyl(ethoxy)phosphoryl]ethyl]tetrahydrofuran-3-yl] acetate (140 mg, 50.68%) as yellow semi-solid.

A solution of[(2R,3R,4R,5R)-4-acetoxy-5-[6-(benzylamino)-2-chloro-purin-9-yl]-2-[2-[diisopropoxyphosphorylmethyl(ethoxy)phosphoryl]ethyl]tetrahydrofuran-3-yl]acetate (140 mg, 188.14 μmol, 1 eq.) in NH₃-MeOH (7M, 1.5 mL, 57.56 eq.)was stirred at room temperature for 2 h. The mixture was concentrated toremove the solvent to afford the crude product(2R,3R,4S,5R)-2-[6-(benzylamino)-2-chloro-purin-9-yl]-5-[2-[diisopropoxyphosphorylmethyl(ethoxy)phosphoryl]ethyl]tetrahydrofuran-3,4-diol asyellow semi-solid. It was used directly for the next step.

To a solution of(2R,3R,4S,5R)-2-[6-(benzylamino)-2-chloro-purin-9-yl]-5-[2-[diisopropoxyphosphorylmethyl(ethoxy)phosphoryl]ethyl]tetrahydrofuran-3,4-diol(124.18 mg, 188.14 μmol, 1 eq.) in DMF (4 mL) was addedbromo(trimethyl)silane (432.05 mg, 2.82 mmol, 15 eq.). The mixture wasstirred at 50° C. for 2 h. Then the mixture was quenched by TEACsolution until pH=8 at 0° C. The mixture was concentrated to dry andadded H₂O (5 mL) and purified by reversed-phase chromatography(C18-column) (H₂O/ACN from 100:0 to 90:10) to get a-1 (20 mg, 19.40%) aswhite solid. ¹H NMR (500 MHz, CD₃OD) δ ppm: 1.88-2.13 (m, 6H), 4.04-4.07(m, 1H), 4.21-4.23 (m, 1H), 4.61-4.63 (m, 1H), 4.77 (s, 2H), 5.94 (d,J=2.5 Hz, 1H), 7.27-7.29 (m, 1H), 7.33-7.36 (m, 2H), 7.41-7.42 (m, 2H),8.30 (s, 1H); ¹³C NMR (125 MHz, CD₃OD) δ ppm: 27.85, 28.11, 28.89,31.15, 31.75, 32.07, 32.66, 45.09, 74.36, 75.62, 86.30, 86.43, 69.41,119.67, 128.33, 128.84, 129.56, 139.98, 141.01, 151.25, 155.72, 156.40;³¹P NMR (203 MHz, CD₃OD) δ ppm:14.99, 36.71; m/z (ESI⁺): 548.0 (M+H).

Example 20. Synthesis of Compound a-9

To a solution of[(2R,3R,4R,5R)-4-acetoxy-5-(2,6-dichloropurin-9-yl)-2-[2-[diisopropoxyphosphorylmethyl(ethoxy)phosphoryl]ethyl]tetrahydrofuran-3-yl]acetate (660 mg, 980.08 μmol, 1 eq.) and[(3R,5S)-3,5-dimethyl-1-adamantyl]ammonium; chloride (317.20 mg, 1.47mmol, 1.5 eq.) in 1,4-dioxane (11 mL) was added DIPEA (380.00 mg, 2.94mmol, 512.12 μL, 3 eq.). The mixture was stirred at 130° C. for 16 h.The reaction completion was detected by TLC (EtOAc:MeOH=10:1). Themixture was concentrated and purified by column chromatography on silicagel eluted with (EA/MeOH from 100:0 to 92:8) to give[(2R,3R,4R,5R)-4-acetoxy-5-[2-chloro-6-[[(3R,5S)-3,5-dimethyl-1-adamantyl]amino]purin-9-yl]-2-[2-[diisopropoxyphosphorylmethyl(ethoxy)phosphoryl]ethyl]tetrahydrofuran-3-yl]acetate(310 mg, 38.75%) as yellow semi-solid. ¹H NMR (500 MHz, CD₃OD): δ ppm:0.90 (s, 6H), 1.13-1.50 (m, 20H), 1.79-2.26 (m, 14H), 2.64 (dd, J=31.9,15.0 Hz, 2H), 4.10 (qd, J=14.4, 7.1 Hz, 2H), 4.64-4.79 (m, 2H),5.50-5.61 (m, 1H), 5.83-5.94 (m, 1H), 6.09 (d, J=4.9 Hz, 1H), 6.99 (s,1H), 8.18 (d, J=6.0 Hz, 1H).

A solution of[(2R,3R,4R,5R)-4-acetoxy-5-[2-chloro-6-[[(3R,5S)-3,5-dimethyl-1-adamantyl]amino]purin-9-yl]-2-[2-[diisopropoxyphosphorylmethyl(ethoxy)phosphoryl]ethyl]tetrahydrofuran-3-yl] acetate (310 mg, 379.78 μmol, 1 eq.) in NH₃-MeOH(7M, 6 mL, 110.59 eq.) was stirred at room temperature for 3 h. Themixture was concentrated to remove the solvent. EtOAc (35 mL) was addedinto the residue and washed by brine. The organic layer was dried withNa₂SO₄ and concentrated to afford the crude product(2R,3R,4S,5R)-2-[2-chloro-6-[[(3R,5S)-3,5-dimethyl-1-adamantyl]amino]purin-9-yl]-5-[2-[diisopropoxyphosphorylmethyl(ethoxy)phosphoryl]ethyl]tetrahydrofuran-3,4-diol(250 mg, 89.91%) as yellow semi-solid. The solid was used directly forthe next step.

To a solution of(2R,3R,4S,5R)-2-[2-chloro-6-[[(3R,5S)-3,5-dimethyl-1-adamantyl]amino]purin-9-yl]-5-[2-[diisopropoxyphosphorylmethyl(ethoxy)phosphoryl]ethyl]tetrahydrofuran-3,4-diol(250 mg, 341.44 μmol, 1 eq.) in DMF (8 mL) was addedbromo(trimethyl)silane (993.18 mg, 6.49 mmol, 856.19 μL, 19 eq.). Themixture was stirred at 50° C. for 2 h. Then the mixture was quenched byTEAC solution until pH=8 at 0° C. The mixture was concentrated to dryand H₂O (8 mL) was added, then compound was purified by reverse-phasechromatography (C18-column) (H₂O/ACN from 100:0 to 80:20) to get a-9(101 mg, 41.02%) as white solid. ¹H NMR (500 MHz, CD₃OD) δ ppm: 0.89 (s,6H), 1.13-1.23 (m, 2H), 1.35 (d, J=12.3 Hz, 2H), 1.45 (d, J=12.0 Hz,2H), 1.81 (d, J=11.8 Hz, 2H), 1.85-2.01 (m, 5H), 2.03-2.15 (m, 5H), 2.18(d, J=2.9 Hz, 1H), 4.02 (dd, J=12.2, 5.3 Hz, 1H), 4.17 (t, J=5.1 Hz,1H), 4.57 (t, J=5.1 Hz, 1H), 5.89 (d, J=4.8 Hz, 1H), 8.26 (s, 1H); ¹³CNMR (125 MHz, CD₃OD) δ ppm: 27.68, 27.94, 28.72, 30.75, 31.69, 33.44,40.72, 43.75, 51.75, 55.91, 74.37, 75.65, 86.15, 86.29, 89.47, 119.70,140.55, 150.78, 154.97, 155.88; ³¹P NMR (203 MHz, CD₃OD) δ ppm:14.98,38.21; m/z (ESI⁺): 620.1 (M+H).

Example 21. Synthesis of Compound a-19

To a solution of[(2R,3R,4R,5R)-4-acetoxy-5-(2,6-dichloropurin-9-yl)-2-[2-[diisopropoxyphosphorylmethyl(ethoxy)phosphoryl]ethyl]tetrahydrofuran-3-yl]acetate (250 mg, 371.24 mol, 1 eq.) and dodecan-1-amine (82.57 mg,445.49 μmol, 1.2 eq.) in 1,4-dioxane (2.5 mL) was added DIPEA (143.94mg, 1.11 mmol, 193.99 μL, 3 eq.). The mixture was stirred at 120° C. for16 h. The reaction completion was detected by TLC (EtOAc:MeOH=10:1). Themixture was concentrated and purified by column chromatography on silicagel eluted with (EA/MeOH from 100:0 to 90:10) to give[(2R,3R,4R,5R)-4-acetoxy-5-[2-chloro-6-(dodecylamino)purin-9-yl]-2-[2-[diisopropoxyphosphorylmethyl(ethoxy)phosphoryl]ethyl]tetrahydrofuran-3-yl] acetate (150 mg, 49.14%) as yellow semi-solid.

To a solution of[(2R,3R,4R,5R)-4-acetoxy-5-[2-chloro-6-(dodecylamino)purin-9-yl]-2-[2-[diisopropoxyphosphorylmethyl(ethoxy)phosphoryl]ethyl]tetrahydrofuran-3-yl]acetate (150 mg, 182.41 μmol, 1 eq.) in NH₃-MeOH (7M, 1.5 mL, 57.56 eq.)was stirred at room temperature for 2 h. The mixture was concentrated toremove the solvent to afford the crude product(2R,3R,4S,5R)-2-[2-chloro-6-(dodecylamino)purin-9-yl]-5-[2-[diisopropoxyphosphorylmethyl(ethoxy)phosphoryl]ethyl]tetrahydrofuran-3,4-diol asyellow semi-solid. It was used directly for the next step.

To a solution of(2R,3R,4S,5R)-2-[2-chloro-6-(dodecylamino)purin-9-yl]-5-[2-[diisopropoxyphosphorylmethyl(ethoxy)phosphoryl]ethyl]tetrahydrofuran-3,4-diol(134.66 mg, 182.41 μmol, 1 eq.) in DMF (4 mL) was addedbromo(trimethyl)silane (418.90 mg, 2.74 mmol, 15 eq.). The mixture wasstirred at 50° C. for 2 h. Then the mixture was quenched by TEACsolution until pH 8 at 0° C. The mixture was concentrated to dry and H₂O(5 mL) was added, then compound was purified by reverse-phasechromatography (C18-column) (H₂O/ACN from 100:0 to 70:30) to get a-19(20 mg, 17.51%) as white solid. ¹H NMR (500 MHz, CD₃OD) δ ppm:0.86-0.089 (m, 3H), 1.26-1.30 (m, 18H), 1.64-1.67 (m, 2H), 1.92-2.17 (m,6H), 1.81 (d, J=11.8 Hz, 2H), 1.85-2.01 (m, 5H), 2.03-2.15 (m, 5H),3.51-3.54 (m, 2H), 4.05-4.06 (d, J=5.0, 1H), 4.19 (t, J=5.0 Hz, 1H),4.59 (t, J=5.0 Hz, 1H), 5.91 (d, J=5.0 Hz, 1H), 8.25 (s, 1H); ¹³C NMR(125 MHz, CD₃OD) δ ppm: 13.13, 22.33, 26.17, 26.45, 28.87, 28.97, 29.04,29.26, 29.32, 31.64, 40.20, 72.95, 74.25, 84.86, 87.88, 118.09, 139.24,149.41, 154.44, 155.17; ³¹P NMR (203 MHz, CD₃OD) δ ppm:15.01, 39.35; m/z(ESI⁺): 626.2 (M+H).

Example 22. Synthesis of Compound a-31

To a solution of[(2R,3R,4R,5R)-4-acetoxy-5-(2,6-dichloropurin-9-yl)-2-[2-[diisopropoxyphosphorylmethyl(ethoxy)phosphoryl]ethyl]tetrahydrofuran-3-yl]acetate (1.5 g, 2.23 mmol, 1 eq) and 2-azaspiro[4.5]decane (465.22 mg,3.34 mmol, 1.5 eq.) in 1,4-dioxane (15 mL) was added DIPEA (1.01 g, 7.80mmol, 1.36 mL, 3.0 eq.). The mixture was stirred at 130° C. for 16 h.The reaction completion was detected by TLC (EA/MeOH=20:1). The mixturewas concentrated and purified by column chromatography on silica geleluted with (EA/MeOH from 100:0 to 95:5) to give[(2R,3R,4R,5R)-4-acetoxy-5-[6-(2-azaspiro[4.5]decan-2-yl)-2-chloro-purin-9-yl]-2-[2-[diisopropoxyphosphorylmethyl(ethoxy)phosphoryl] ethyl] tetrahydrofuran-3-yl] acetate (950 mg, 54.95%). 1HNMR (500 MHz, CD3OD): δ ppm 1.26-1.36 (m, 17H), 1.51-1.53 (m, 2H),1.83-2.17 (m, 12H), 2.62-2.69 (m, 2H), 3.39-3.53 (m, 1H), 3.71-3.73 (m,1H), 3.93-3.95 (m, 1H), 4.13-4.27 (m, 4H), 4.69-4.71 (m, 2H), 5.58-5.59(m, 1H), 5.91-5.93 (m, 1H), 6.10-6.12 (m, 1H), 6.15-6.17 (m, 1H).

A solution of[(2R,3R,4R,5R)-4-acetoxy-5-[6-(2-azaspiro[4.5]decan-2-yl)-2-chloro-purin-9-yl]-2-[2-[diisopropoxyphosphorylmethyl(ethoxy)phosphoryl]ethyl]tetrahydrofuran-3-yl]acetate (950 mg, 1.22 mmol, 1 eq.) in NH3-MeOH (7 M, 10 mL, 57.19 eq.)was prepared. The mixture was stirred at room temperature for 16 h. Themixture was concentrated and EtOAc (30 mL) was added. The organic layerwas washed with NaCl aqueous, dried (Na2SO4), filtered, and evaporatedto dryness to give(2R,3R,4S,5R)-2-[6-(2-azaspiro[4.5]decan-2-yl)-2-chloro-purin-9-yl]-5-[2-[diisopropoxyphosphorylmethyl(ethoxy)phosphoryl]ethyl]tetrahydrofuran-3,4-diol (650 mg, 76.73%). 1HNMR (500 MHz, CD3OD): δ ppm 1.27-1.36 (m, 15H), 1.52-1.54 (m, 10H),1.84-1.94 (m, 2H), 2.05-2.18 (m, 4H), 2.61-2.68 (m, 2H), 3.52-3.54 (m,1H), 3.72-3.74 (m, 1H), 3.88-4.03 (m, 2H), 4.12-4.14 (m, 3H), 4.27-4.29(m, 1H), 4.62-4.73 (m, 3H), 5.89 (s, 1H), 8.14-8.15 (m, 1H).

To a solution of(2R,3R,4S,5R)-2-[6-(2-azaspiro[4.5]decan-2-yl)-2-chloro-purin-9-yl]-5-[2-[diisopropoxyphosphorylmethyl(ethoxy)phosphoryl]ethyl]tetrahydrofuran-3,4-diol(100 mg, 144.48 μmol, 1 eq.) in DMF (4 mL) was addedbromo(trimethyl)silane (331.79 mg, 2.17 mmol, 15 eq.) at 0° C. Themixture was stirred at 50° C. for 2 h. Then the mixture was quenched byTEAC solution until pH 8 at 0° C. The mixture was concentrated to dryand H₂O (10 mL) was added, and purified by reverse-phase chromatography(C18-column) (H₂O/ACN from 100:0 to 90:10) to get desired product a-31(20 mg, 20.32%). 1H NMR (CD3OD, 500 MHz): δ ppm 1.51-1.57 (m, 10H),1.83-2.10 (m, 8H), 3.51-3.53 (m, 1H), 3.71-3.73 (m, 1H), 3.95-3.97 (m,1H), 4.03-4.04 (m, 1H), 4.16-4.19 (m, 2H), 4.57-4.59 (m, 1H), 5.90-5.91(d, 1H), 8.19-8.20 (m, 1H); ¹³C NMR (CD3OD, 125 MHz): δ ppm 23.02,25.87, 26.18, 26.40, 27.18, 29.21, 29.80, 30.13, 30.73, 34.89, 40.37,42.67, 72.94, 74.05, 84.70, 87.93, 118.71, 138.65, 150.96, 153.21,153.83; 31P NMR (CD3OD, 203 MHz): δ 15.12, 38.73; m/z (ESI+): 580.1(M+H).

Example 23. Synthesis of Compound c-13

To a mixture of(3aR,4R,6R,6aR)-6-(2-diethoxyphosphorylethyl)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxole(2.50 g, 7.39 mmol, 1 eq.) in 1,4-dioxane (10 mL) was added 1 N H₂SO₄ (1M, 25 mL, 3.38 eq), and it was heated to reflux for 2.5 h. Then the pHwas adjusted to 7 by aq. NaHCO₃ (50 mL), and it was concentrated todryness. To the residue in Pyridine (21 mL) was added Ac₂O (836.84 mg,8.20 mmol, 4.2 mL), and it was stirred at room temperature for 16 h. Theresulting solution was evaporated to dryness. The residue was purifiedby column chromatography on silica gel eluted with (DCM/MeOH from 100:0to 95:05) to give[(2R,3R,4R)-4,5-diacetoxy-2-(2-diethoxyphosphorylethyl)tetrahydrofuran-3-yl] acetate (1.9 g, 62.67%). ¹H NMR (500 MHz, CDCl3):δ ppm 1.36-1.54 (m, 6H), 1.81-2.30 (m, 13H), 1.94-2.05 (m, 2H),4.13-4.31 (m, 5H), 4.90-5.23 (m, 2H), 6.16-6.41 (m, 1H).

To a solution of [(2R,3R,4R)-4,5-diacetoxy-2-(2-diethoxyphosphorylethyl)tetrahydrofuran-3-yl] acetate (1.9 g, 4.63 mmol, 1 eq) in ACN (19 mL)was added DBU (775.39 mg, 5.09 mmol, 760.18 μL, 1.1 eq) and TMSOTf (2.26g, 10.19 mmol, 1.84 mL, 2.2 eq) at −10° C. The mixture was stirred at50° C. for 1 h. The mixture was concentrated and purified by columnchromatography on silica gel eluted with (EA/MeOH from 100:0 to 95:5) togive[(2R,3R,4R,5R)-4-acetoxy-5-(2,6-dichloropurin-9-yl)-2-(2-diethoxyphosphorylethyl) tetrahydrofuran-3-yl] acetate (1.3 g, 52.06%). ¹H NMR (500 MHz,CDCl₃): δ ppm 1.24-1.34 (m, 6H), 2.01-2.14 (m, 10H), 4.09-4.12 (m, 5H),5.56-5.58 (m, 1H), 5.88-5.90 (m, 1H), 6.24-6.25 (d, 1H), 8.68 (s, 1H).

To a solution of[(2R,3R,4R,5R)-4-acetoxy-5-(2,6-dichloropurin-9-yl)-2-(2-diethoxyphosphorylethyl)tetrahydrofuran-3-yl]acetate (1.3 g, 2.41 mmol, 1 eq.) and[(3R,5S)-3,5-dimethyl-1-adamantyl]ammonium; chloride (624.12 mg, 2.89mmol, 1.2 eq.) in 1,4-dioxane (13 mL) was added DIPEA (934.61 mg, 7.23mmol, 1.26 mL, 3 eq.). The mixture was stirred at 130° C. for 16 h. Thereaction completion was detected by TLC. The mixture was concentratedand purified by column chromatography on silica gel eluted with (EA/MeOHfrom 100:0 to 90:10) to give[(2R,3R,4R,5R)-4-acetoxy-5-[2-chloro-6-[[(3R,5S)-3,5-dimethyl-1-adamantyl]amino]purin-9-yl]-2-(2-diethoxyphosphorylethyl)tetrahydrofuran-3-yl]acetate (700 mg, 42.57%). ¹H NMR (500 MHz, CD₃OD): δ ppm 0.84-0.91 (m,6H), 1.20-1.32 (m, 9H), 1.36-1.52 (m, 4H), 1.82-2.01 (m, 6H), 2.07-2.14(m, 10H), 4.08-4.11 (m, 5H), 5.57-5.60 (m, 1H), 5.87-5.89 (m, 1H),6.09-6.10 (m, 1H), 8.16 (m, 1H).

To a solution of[(2R,3R,4R,5R)-4-acetoxy-5-[2-chloro-6-[[(3R,5S)-3,5-dimethyl-1-adamantyl]amino]purin-9-yl]-2-(2-diethoxyphosphorylethyl)tetrahydrofuran-3-yl]acetate(700 mg, 1.03 mmol, 1 eq) was added NH₃-MeOH (7 M, 7 mL, 47.75 eq.). Themixture was stirred at room temperature for 16 h. The mixture wasconcentrated and EtOAc (30 mL) was added, and the organic layer waswashed with aq. NaCl, dried (Na₂SO₄), filtered, and evaporated to give(2R,3R,4S,5R)-2-[2-chloro-6-[[(3R,5S)-3,5-dimethyl-1-adamantyl]amino]purin-9-yl]-5-(2-diethoxyphosphorylethyl)tetrahydrofuran-3,4-diol (400 mg, 65.18%). ¹H NMR (500 MHz, CD₃OD): δppm 0.90-0.92 (m, 6H), 1.20-1.26 (m, 2H), 1.29-1.32 (m, 6H), 1.36-1.39(m, 2H), 1.46-1.48 (m, 2H), 1.82-1.84 (m, 2H), 1.91-1.93 (m, 3H),1.96-2.05 (m, 3H), 2.10-2.12 (m, 2H), 2.20-2.21 (m, 1H), 4.03-4.11 (m,5H), 4.27-4.29 (m, 1H), 4.69-4.71 (m, 1H), 5.86-5.87 (m, 1H), 8.15 (s,1H).

To a solution of(2R,3R,4S,5R)-2-[2-chloro-6-[[(3R,5S)-3,5-dimethyl-1-adamantyl] amino]purin-9-yl]-5-(2-diethoxyphosphorylethyl)tetrahydrofuran-3,4-diol (100mg, 167.20 μmol, 1 eq) in DMF (4 mL) was added bromo(trimethyl)silane(383.97 mg, 2.51 mmol, 331.01 μL, 15 eq) at 0° C. The mixture wasstirred at 50° C. for 2 h. Then the mixture was quenched by TEACsolution until pH 8 at 0° C. The mixture was concentrated and H₂O (10mL) was added, then compound was purified by reverse-phasechromatography (C18-column) (H₂O/ACN from 100:0 to 70:30) to get c-13(40 mg, 37.20%). ¹H NMR (CD₃OD, 500 MHz): δ ppm 1.19-1.26 (m, 2H),1.36-1.38 (m, 2H), 1.45-1.48 (m, 2H), 1.63-1.74 (m, 2H), 1.82-1.84 (m,2H), 1.90-1.95 (m, 2H), 1.98-2.10 (m, 4H), 2.19-2.21 (m, 1H), 4.02-4.03(m, 1H), 4.15-4.17 (m, 1H), 4.55-4.56 (m, 1H), 5.90-5.91 (d, 1H), 8.22(s, 1H). ¹³C NMR (CD3OD, 125 MHz): δ ppm 25.41, 26.49, 29.21, 30.75,31.70, 33.45, 40.71, 43.76, 51.76, 55.91, 74.60, 75.76, 85.99, 86.13,89.71, 119.74, 140.42, 150.68, 154.98, 155.88; ³¹P NMR (CD3OD, 203 MHz):δ ppm 24.12; m/z (ESI⁻): 540.2 (M−H).

Biological Assays

1. CD73 Inhibitor Screening Assay

To evaluate the inhibitory effect of the compounds on CD73, MalachiteGreen Phosphate Detection Kit (R&D, Cat #DY996) was used. Briefly,compounds were dissolved and diluted to the desired concentration usingphosphate-free buffer (Tris-HCl, pH 7.3). 25 μL of the compound solutionwas added to an equal volume of CD73 protein solution (2× concentration,0.5 μg/mL, Novoprotein, Cat #C446), followed by a 5-minute incubation atroom temperature. 10 μL of Malachite Green Reagent A was added to eachwell, mixed thoroughly and incubated for 10 minutes at room temperature.10 μL of Malachite Green Reagent B was then added to each well, mixedthoroughly and incubated for 20 minutes at room temperature. Finally,the optical density of each well was determined using a microplatereader set to 620 nm.

The inhibitory activity of selected compounds is given in Table 2. Inaddition, compounds such as a-1, a-9, and a-31 showed inhibitoryactivity in the assay.

TABLE 2 Compound inhibitory activity in CD73 enzyme assay. Compound No.Potency¹ 1 +++ 2 ++ 6 +++ 7 +++ 8 ++ 9 +++ 10 ++ 11 ++ 12 ++ 15 +++ 16++ 17 +++ 18 ++ 19 + 20 ++ 22 +++ 23 ++ 31 +++ 51 +++ ¹“+” denotesIC₅₀ >100 nM; “++” denotes IC₅₀ of 10-100 nM; “+++” denotes IC₅₀ <10 nM.2. Cell-Based Assay of CD73 Activity

Prepare 5× compound solution by dissolving and diluting the compounds todesired concentrations using serum free RMPI-1640 medium containing 1 μMAMP. A375 cells are collected and washed twice with PBS, then suspendedin serum-free RMPI-1640 medium to a density of 1.125×10⁵/mL. Aliquot 80μL of the cell suspension to a 96-well plate, then add 20 μL of the 5×compound solution, mix gently and culture for 16 hrs at 37° C., 5% CO₂.After incubation, transfer 50 μL of supernatant from each well to a new96-well plate. Then sequentially add 2 μL of 2.5 μM ATP and 50 μL ofCelltiter Glo reagent to each well. Measure luminescence using PheraStar(BMG).

3. Pharmacokinetic Evaluation of the Compounds

Following a single i.v. (1 mg/kg) or i.g. (3 mg/kg) administration ofthe compounds to fasted male SD rats, blood samples were collected at0.08, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h after administration. Plasma wasseparated by centrifugation (8000 rpm) and frozen (−20° C.) until thesample was analyzed. Concentrations of the compounds in rat plasma weredetermined by HPLC-MS/MS. Plasma was dispensed into appropriate tubescontaining internal standard and methanol or acetonitrile. The tubeswere mixed vigorously for 3 minutes to achieve deproteinization and thencentrifuged at 8000 rpm for 5 minutes. The supernatant was transferredto an autosampler vial, and was injected into the chromatographicsystem. Pharmacokinetic Parameters including AUC_(0-t), C max, t maxt_(1/2), MRT, Cl and Vd were calculated using WinNonlin 6.3 software.Absolute bioavailability was calculated as follows:F=[AUC(i.g.)*dose(i.v.)]/[AUC(i.v.)*dose(i.g.)]*100%.

Although this invention is described in detail with reference toembodiments thereof, these embodiments are offered to illustrate but notto limit the invention. It is possible to make other embodiments thatemploy the principles of the invention and that fall within its spiritand scope as defined by the claims appended hereto.

What is claimed is:
 1. A compound of Formula I, or a pharmaceuticallyacceptable ester or salt thereof:

where: W is oxygen, sulfur, nitrogen, or a methylene group; X is amoiety selected from phosphonyl (—P(═O)(OR)—), sulfonyl (—S(═O)₂—), andcarbonyl (—C(═O)—), where R is a hydrogen, an ester-forming group, or aprotecting group; or X and W together form —(CR⁷R⁸)_(n), where n is aninteger from 0 to 3, and R⁷ and R⁸ are independently selected from ahydrogen, a halogen, a hydroxyl group, and a lower alkyl group having 1to 4 carbon atoms; Y is selected from phosphonate (—PO₃R₂), sulfonate(—SO₃R), and carboxylate (—CO₂R), where R is a hydrogen, anester-forming group, or a protecting group; R¹ is a hydroxyl group or ahydrogen; R² is a chlorine or a hydrogen; and R³ and R⁴ areindependently selected from a hydrogen, an alkyl group, an alkenyl groupand an alkynyl group, where at least one of R³ and R⁴ has 1 to 30 carbonatoms, wherein at least one of R³ and R⁴ has 11 to 30 carbon atoms whenW is O or S; or R³ and R⁴ are independently selected from a hydrogen anda ring system which is a monocycle, bicycle, tricycle, spiral-ring,fused-ring, or bridged-ring containing carbocyclic or heterocyclic ringsystem, the carbocyclic ring system being aromatic or non-aromatic,provided that R³ and R⁴ are not both hydrogen, and provided that thering system is not a monocycle when W is O or S; or R³, R⁴, and thenitrogen atom to which they are attached form a heterocyclic systemwhich is independently selected from a monocycle, a bicycle, a tricycle,a spiral-ring, a fused-ring, and a bridged-ring system.
 2. The compoundof claim 1, wherein R³ is a hydrogen or a lower alkyl; and R⁴ is—C(═O)R⁵, —C(═O)NHR⁵ or —C(═O)OR⁵, where R⁵ is a C₁₋₃₀ alkyl group, aC₂₋₃₀ alkenyl group or a C₂₋₃₀ alkynyl group, provided that R⁵ is aC₁₁₋₃₀ alkyl, C₁₁₋₃₀ alkenyl or C₁₁₋₃₀ alkynyl group when W is O or S.3. The compound of claim 1, wherein R³ is a hydrogen or a lower alkyl;and R⁴ is —C(═O)R⁵, —C(═O)NHR⁵ or —C(═O)OR⁵, where R⁵ is a ring systemhaving a monocycle, a bicycle, a tricycle, a spiral-ring, a fused-ringor a bridged-ring containing a carbocyclic or a heterocyclic ringsystem, the carbocyclic ring system being aromatic or non-aromatic, andthe heterocyclic ring system being substituted or unsubstituted,provided that R⁵ is not a monocycle ring system when W is O or S.
 4. Thecompound of claim 1, wherein the compound is a compound of Formula IVa,Formula IVb, Formula IVc, Formula Va, Formula VIa, Formula VIIa, orFormula VIIIa, or a pharmaceutically acceptable salt or ester thereof:

where: in formula IVc, n is an integer from 0 to 3, and R⁷ and R⁸ areindependently selected from a hydrogen, a halogen, a hydroxyl group anda lower alkyl group which has from 1 to 4 carbon atoms; and in formulaVIIa and formula VIIIa, R is a hydrogen, an ester-forming group, or aprotecting group.
 5. The compound of claim 1, wherein the compound is acompound of Formula IXa, or a pharmaceutically acceptable salt or esterthereof:

where: R is a hydrogen, an ester-forming group, or a protecting group;R¹ is a hydroxyl group or a hydrogen; R² is a hydrogen or a chlorine;and R³ is a hydrogen or a lower alkyl, and R⁴ is an alkyl, an alkenyl,or an alkynyl group having 1 to 30 carbon atoms; or R³ is a hydrogen ora lower alkyl, and R⁴ is a substituent group containing a monocyclic,bicyclic, tricyclic, or multi-cyclic ring system, where the ring systemis fused, spiral, bridged, or parallel, and the ring system iscarbocyclic, aliphatic, aromatic, heterocyclic, or a combinationthereof; or R³ is a hydrogen or a lower alkyl, and R⁴ is —C(═O)R⁵,—C(═O)NHR⁵ or —C(═O)OR⁵, where R⁵ is an alkyl group, an alkenyl group oran alkynyl group having 1 to 30 carbon atoms; or R³ is a hydrogen or alower alkyl, and R⁴ is —C(═O)R⁵ or —C(═O)OR⁵, where R⁵ is a substituentgroup containing a bicycle, tricycle, spiral-ring, fused-ring orbridged-ring containing a carbocyclic or a heterocyclic ring system, thecarbocyclic ring system being aromatic or non-aromatic, the heterocyclicring system being substituted or unsubstituted), where the ring iscarbocyclic, aliphatic, aromatic, heterocyclic, or a combinationthereof; or R³ is a hydrogen or a lower alkyl, and R⁴ is anunsubstituted or substituted 1-adamantyl, α-naphthylmethyl, orβ-naphthylmethyl; or R³, R⁴, and the nitrogen atom to which they areattached form a heterocyclic system independently selected from amonocycle, a bicycle, a tricycle, a spiral-ring, a fused-ring, and abridged-ring.
 6. The compound of claim 5 wherein R³ is a hydrogen or alower alkyl; and R⁴ is a group containing an adamantyl moiety.
 7. Thecompound of claim 6, wherein R⁴ is substituted or non-substituted1-adamantyl; substituted or non-substituted 2-adamantyl; substituted ornon-substituted 1-adamantylmethyl; substituted or non-substituted1-adamantylethyl; substituted or non-substituted 1-adamantylpropyl; orsubstituted or non-substituted 1-adamantylbutyl.
 8. The compound ofclaim 5, wherein R³ is a hydrogen or a lower alkyl; and R⁴ is a groupcontaining a naphthyl moiety.
 9. The compound of claim 8, wherein R⁴ issubstituted or non-substituted α-naphthyl; substituted ornon-substituted β-naphthyl; substituted or non-substitutedα-naphthylmethyl; substituted or non-substituted β-naphthylmethyl;substituted or non-substituted naphthylethyl; substituted ornon-substituted naphthylpropyl; or substituted or non-substitutednaphthylbutyl.
 10. The compound of claim 1, wherein the C, H, O, and Natoms in the compound are each independently selected from atoms ofnatural abundance and isotope-enriched atoms, wherein theisotope-enriched atoms are selected from ¹²C, ¹³C, and ¹⁴C for carbon;selected from ¹H, ²H, and ³H for hydrogen; selected from ¹⁶O, ¹⁷O, and¹⁸O for oxygen; and selected from ¹⁴N and ¹⁵N for nitrogen.
 11. Apharmaceutical composition comprising the compound or pharmaceuticallyacceptable salt or ester thereof of claim 1 and a pharmaceuticallyacceptable carrier.
 12. A method of treating or preventing aCD73-associated disease, disorder or condition in a subject in needthereof, comprising administering a therapeutically effective amount ofthe compound of claim 1 to the subject, such that the CD73-associateddisease, disorder or condition is treated or prevented in the subject,wherein the CD73-associated disease, disorder or condition isCD73-mediated immunosuppression; cancer; or an immune-related disease,disorder or condition selected from the group consisting of rheumatoidarthritis, kidney failure, lupus, asthma, psoriasis, colitis,pancreatitis, allergies, fibrosis, anemia fibromyalgia, Alzheimer'sdisease, congestive heart failure, stroke, aortic valve stenosis,arteriosclerosis, osteoporosis, Parkinson's disease, infections, Crohn'sdisease, ulcerative colitis, allergic contact dermatitis, eczema,systemic sclerosis and multiple sclerosis.
 13. The method of claim 12,wherein said compound is administered in an amount effective to reverse,slow or stop the progression of CD73-mediated immunosuppression in thesubject.
 14. The method of claim 12, wherein the CD73-associateddisease, disorder or condition is cancer.
 15. The method of claim 14,wherein said cancer is a cancer of the prostate, colon, rectum,pancreas, cervix, stomach, endometrium, brain, liver, bladder, ovary,testis, head, neck, skin, mesothelial lining, white blood cell,esophagus, breast, muscle, connective tissue, lung, adrenal gland,thyroid, kidney, or bone.
 16. The method of claim 12, wherein theCD73-associated disease, disorder or condition is an immune-relateddisease, disorder or condition selected from the group consisting ofrheumatoid arthritis, kidney failure, lupus, asthma, psoriasis, colitis,pancreatitis, allergies, fibrosis, anemia fibromyalgia, Alzheimer'sdisease, congestive heart failure, stroke, aortic valve stenosis,arteriosclerosis, osteoporosis, Parkinson's disease, infections, Crohn'sdisease, ulcerative colitis, allergic contact dermatitis, eczema,systemic sclerosis and multiple sclerosis.
 17. A method of treatingcancer in a subject, comprising administering to said subject aneffective amount of the compound of claim 1 and an immune checkpointinhibitor, such that cancer is treated in the subject.
 18. The method ofclaim 17, wherein said administering is prior to, concurrent with, orsubsequent to, radiation treatment.
 19. The method of claim 17, whereinsaid compound and said immune checkpoint inhibitor are administered incombination or sequentially.
 20. The method of claim 17, wherein saidimmune checkpoint inhibitor is selected from the group consisting ofipulimumab, nivolumab and lambrolizumab.
 21. A compound which is:

or a pharmaceutically acceptable salt or ester thereof.